Paramagnetic Chemical Probes for Studying Biological Macromolecules

Abstract

Paramagnetic chemical probes have been used in electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopy for more than four decades. Recent years witnessed a great increase in the variety of probes for the study of biological macromolecules (proteins, nucleic acids, and oligosaccharides). This Review aims to provide a comprehensive overview of the existing paramagnetic chemical probes, including chemical synthetic approaches, functional properties, and selected applications. Recent developments have seen, in particular, a rapid expansion of the range of lanthanoid probes with anisotropic magnetic susceptibilities for the generation of structural restraints based on residual dipolar couplings and pseudocontact shifts in solution and solid state NMR spectroscopy, mostly for protein studies. Also many new isotropic paramagnetic probes, suitable for NMR measurements of paramagnetic relaxation enhancements, as well as EPR spectroscopic studies (in particular double resonance techniques) have been developed and employed to investigate biological macromolecules. Notwithstanding the large number of reported probes, only few have found broad application and further development of probes for dedicated applications is foreseen.

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Paramagnetic Chemical Probes for Studying Biological
Macromolecules
Qing Miao, Christoph Nitsche, Henry Orton, Mark Overhand, Gottfried Otting, and Marcellus Ubbink*
Cite This: https://doi.org/10.1021/acs.chemrev.1c00708
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ABSTRACT: Paramagnetic chemical probes have been used in electron paramagnetic
resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopy for more than four
decades. Recent years witnessed a great increase in the variety of probes for the study of
biological macromolecules (proteins, nucleic acids, and oligosaccharides). This Review aims
to provide a comprehensive overview of the existing paramagnetic chemical probes, including
chemical synthetic approaches, functional properties, and selected applications. Recent
developments have seen, in particular, a rapid expansion of the range of lanthanoid probes
with anisotropic magnetic susceptibilities for the generation of structural restraints based on
residual dipolar couplings and pseudocontact shifts in solution and solid state NMR
spectroscopy, mostly for protein studies. Also many new isotropic paramagnetic probes,
suitable for NMR measurements of paramagnetic relaxation enhancements, as well as EPR
spectroscopic studies (in particular double resonance techniques) have been developed and
employed to investigate biological macromolecules. Notwithstanding the large number of reported probes, only few have found
broad application and further development of probes for dedicated applications is foreseen.
CONTENTS
1. Paramagnetic NMR for Biomolecular Studies B
1.1. General Introduction B
1.2. Qualitative Description of Paramagnetic
Effects in NMR Spectroscopy C
1.3. Quantitative Description of Paramagnetic
Effects in NMR Spectroscopy D
1.3.1. Pseudocontact Shift (PCS) E
1.3.2. Residual Dipolar Coupling (RDC) E
1.3.3. Residual anisotropic chemical shift
(RACS) F
1.3.4. Residual Anisotropic Dipolar Shift
(RADS) F
1.3.5. Saturation Effect F
1.3.6. Paramagnetic Relaxation Enhancement
(PRE) F
1.3.7. Cross-correlated Relaxation (CCR) G
1.4. Paramagnetic NMR of Biomolecules in the
Solid State H
1.4.1. Paramagnetic NMR Effects in Static and
Rotating Solids H
2. Sources of Paramagnetism H
2.1. 3d Block Transition Metal Ions H
2.2. Lanthanoid Ions I
2.3. Nitroxide Radicals J
3. General Overview of Natural and Chemically
Generated Paramagnetic Centers J
3.1. Metalloproteins K
3.1.1. Paramagnetic Metalloproteins K
3.1.2. Metalloproteins with Paramagnetic Sub-
stitution K
3.2. Genetically Encoded Metal Binding Sites K
3.2.1. Natural Amino Acids or Peptides K
3.2.2. Noncanonical Amino Acid and Their
General Synthetic Approaches L
3.3. Synthetic Tags for Proteins M
3.3.1. Nitroxide Probes M
3.3.2. Aminopoly(Carboxylic Acid)-Based
Probes R
3.3.3. Cyclen-Based Tags S
3.3.4. Small Molecule Tags Y
3.3.5. Cosolute Paramagnetic Probes Z
3.3.6. Development of Cysteine Based Probe
Attachment Chemistry AB
3.4. Paramagnetic Probes for DNA and RNA AB
3.4.1. Noncovalent Spin Labels AC
3.4.2. Probe Attachment to the Sugar−Phos-
phate Backbone AD
3.4.3. Probe Attachment to Modified Bases AG
Special Issue: Biomolecular NMR Spectroscopy
Received: August 12, 2021
Reviewpubs.acs.org/CR
© XXXX The Authors. Published by
American Chemical Society A
https://doi.org/10.1021/acs.chemrev.1c00708
Chem. Rev. XXXX, XXX, XXX−XXX
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3.4.5. Fully Synthetic Paramagnetic Nucleo-
tide Probes AI
3.5. Paramagnetic Probes for Oligosaccharides AL
4. Complications in PRE-to-Distance Conversion AM
5. Examples of Applications of Paramagnetic NMR in
Protein Studies AM
5.1. Protein Structure Studies AM
5.1.1. NMR Resonance Assignments in the
Paramagnetic and Diamagnetic States AM
5.1.2. Protein Structure Determination AN
5.1.3. Protein−Protein Interactions AO
5.1.4. Protein−Ligand Interactions AP
5.1.5. Structures of Minor States AQ
5.2. Biomolecules with Paramagnetic Tags in the
Solid State AT
6. Applications of Different Types of Paramagnetic
Probes AT
6.1. Applications of Nitroxide Probes AU
6.2. Applications of Aminopoly(Carboxylic Acid)-
Based Probes AU
6.3. Applications of Cyclen-Based Probes AU
6.3.1. Double-Anchored Probes AU
6.3.2. Applications of Single-Arm Cyclen
Probes AV
6.4. Applications of Small Chemical Probes AW
6.5. Applications of Cosolute Paramagnetic
Probes AW
7. Conclusions and Prospects AX
Author Information AX
Corresponding Author AX
Authors AX
Notes AY
Biographies AY
Acknowledgments AY
Abbreviations AY
Symbols AZ
References BA
1. PARAMAGNETIC NMR FOR BIOMOLECULAR
STUDIES
1.1. General Introduction
Nuclear magnetic resonance (NMR) spectroscopy is the most
widely used spectroscopic technique to obtain structural
information with atomic resolution. The NMR observable
most frequently used for structure determination is the nuclear
Overhauser effect (NOE), which measures dipolar interactions
between protons separated by up to about 5 Å and, thus,
provides internuclear distance restraints.
1,2
Owing to the much
larger magnetic moment of unpaired electrons compared with
nuclei (about 658 times greater for a lone electron than for a
proton), dipolar interactions involving unpaired electrons, such
as in metalloproteins containing paramagnetic metal ions, can be
detected over a much greater distance range. As the para-
magnetic effects of unpaired electrons on the NMR spectrum
tend to be very large and are readily detected, potentially over
distances up to 100 Å from the paramagnetic center, they
present valuable long-range structural information. In fact, 50
years ago and prior to the invention of two-dimensional NMR
spectroscopy, paramagnetic effects were thought to be the key to
3D structure determinations of proteins and other biomolecules
in solution.
3,4
The paramagnetic effects are in many instances
anisotropic, contributing not only distance but also orientational
restraints.
Apart from uses in NMR spectroscopy, paramagnetic labels
also feature most prominently in electron paramagnetic
resonance (EPR) spectroscopy, where they have gained in
importance by their capacity to accurately measure distances
between paramagnetic centers on the nanometer scale. This
Review will focus on applications in NMR spectroscopy, while
also acknowledging the performance of tags for distance
measurements by EPR spectroscopy, specifically in double-
electron−electron resonance (DEER) experiments. Further-
more, europium(III) and terbium(III) are paramagnetic metal
ions which, in conjunction with an aromatic antenna group, can
yield intense luminescence suitable for distance measurements
by Förster resonance energy transfer (FRET).
5,6
The same
probes can thus be suitable for NMR and FRET applications,
but luminescence applications are not discussed in this Review
which focuses on paramagnetism and NMR spectroscopy.
The field of paramagnetic NMR has been reviewed before. In
particular, the theory of paramagnetic NMR and the resulting
spectral effects have been described in many reviews and books,
covering magnetic susceptibility,
7−11
relaxation,
12
and effects in
solids
13
in great detail. This Review presents an integrated
formalism of the various effects and includes the most recent
effects reported.
Initially, paramagnetic NMR spectroscopy of biological
macromolecules took advantage of paramagnetic metal ions
present in proteins,
14,15
and this approach is still finding
application for the study of metalloproteins and also in research
into metal trafficking.
16
Also metal substitution has been used
extensively.
17
With the arrival of paramagnetic tags, the
application of the tool box of paramagnetic NMR spectroscopy
became available for biomolecules that lack a natural para-
magnetic center, expanding its potential greatly. Several reviews
give a general overview of the possibilities of employing
pseudocontact shifts (PCSs), paramagnetic relaxation enhance-
ments (PREs), and residual dipolar couplings (RDCs) (terms to
be explained below) or focus on the range of different tags
available and their applications.
18−26
Specific applications have been the topic of dedicated reviews.
One example is the use of paramagnetism for partial alignment
to generate RDCs for the study of structure and dynamics.
27
PCSs provide useful data for the structure determination of
proteins and protein−protein or protein−ligand complexes, as
sole restraints, or in combination with other data, such as X-ray
diffraction and small-angle X-ray scattering.
10,26,28−30
PREs are
useful in particular because of their ability to report on minor,
“invisible”or “dark”states present in a sample.
31−34
PREs
generated by soluble probes, that is, paramagnetic molecules not
covalently linked to the molecule of interest, are referred to as
solvent PREs and can provide information on molecular surfaces
and changes in surface exposure upon complex formation.
35,36
A
recent review describes the use of paramagnetic NMR in drug
discovery.
37
Tags have been mostly designed for and applied to
proteins, but applications to other biomolecules have started to
appear, such as oligosaccharides,
38,39
and a significant body of
work exists describing oligonucleotides with paramagnetic
centers for EPR measurements.
40−47
The main aim of this Review is to describe the state-of-the-art
of paramagnetic tags for biological macromolecules. We wish to
illustrate the diversity of tags and discuss their pros and cons for
various applications. Contrary to most other reviews, particular
attention is paid to the chemical synthesis of the tags because the
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chemistry of the more advanced cyclen-based lanthanoid tags is
not trivial and in some cases a limiting factor, due to demanding
synthetic routes or limited chemical stability under physiological
conditions and in the presence of proteins. Paramagnetic
compounds have many more applications, e.g., as contrast
agents in magnetic resonance imaging (MRI) and polarizing
agents for dynamic nuclear polarization (DNP). These topics
are considered outside the scope of this Review, and the reader is
referred to excellent, recently published reviews.
48,49
Para-
magnetic tags attached to proteins exclusively for the purpose of
studies by EPR spectroscopy have been comprehensively
discussed previously.
50
Similarly, our discussion omits EPR
spin labels attached via a long and flexible tether, such as
resulting from tagging noncanonical amino acids.
51
In contrast,
our account attempts to give a comprehensive overview over
paramagnetic tags designed specifically for structure studies of
deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)
oligomers by NMR and EPR spectroscopy. Finally, various
paramagnetic compounds have been devised as sample additives
to enhance longitudinal relaxation rates in solid-state NMR
spectroscopy.
52,53
As far as these compounds are not chemically
attached to biological macromolecules, the reader is again
referred to a recent review for a more complete compilation.
54
As paramagnetic probes elicit a multitude of effects depending
on their chemical, physical, and dynamic properties, an
increasing number of probes have been synthesized with the
aim of facilitating their installation in biological macromolecules
and maximizing the information content that can be gathered
from the observation of the paramagnetic effects. About half of
the published paramagnetic probes were described for the first
time in the past five years. To assess their utility for different
purposes, it is necessary to understand the origin and
manifestation of the paramagnetic effects. Many of the most
stringent tag requirements stem from applications in para-
magnetic NMR spectroscopy.
The terms spin label, paramagnetic tag and paramagnetic
probe are frequently found in the literature and also used
throughout this Review. They all refer to paramagnetic
compounds that are introduced and used to generate para-
magnetic effects to study a system of interest. In general, these
terms can be considered synonyms. The term “spin label”is
probably the oldest and has been widely used in the field of EPR
for many years. It refers to labeling a system with an unpaired
electron spin. “Paramagnetic tag”emphasizes the quality of a
small label to give information about the bearer, that is, provide
site-specific information about the tagged molecule. The term
“paramagnetic probe”relates more generally to the function,
that is, probing the molecule under investigation and includes
also soluble compounds that are not bound to specific sites.
1.2. Qualitative Description of Paramagnetic Effects in NMR
Spectroscopy
Paramagnetic effects detected in NMR spectra depend on the
dipolar fields generated by unpaired electrons. If the electron
spins relax slowly compared with the rotational correlation time
of the electron−nucleus vector, the dipolar field of the electron
at the site of the nuclear spin averages to zero, provided that the
molecule reorientates isotropically in solution. In this case, the
chemical shift of the nuclear spin does not change, but the time
fluctuation of the dipolar field can greatly enhance the nuclear
relaxation. Electron spins that relax rapidly compared with the
rotational correlation time of the electron−nucleus vector lead
to a Curie spin, which is the time-averaged net magnetic
moment of the electron spin aligned with the external magnetic
field. The Curie spin constitutes a molecular magnetic
susceptibility. In this situation, it is useful to describe the
magnetic susceptibility associated with the paramagnetic center,
χ, by a tensor that defines its magnitude as a function of the
molecular orientation in the external magnetic field. In general,
the χtensor of a paramagnetic center with rapidly relaxing
electrons is anisotropic and its anisotropic component is
commonly referred to as Δχtensor.
Chemical shift changes observed in NMR spectra due to the
presence of a paramagnetic center are referred to as hyperfine
shifts. For paramagnetic centers with a Curie spin, hyperfine
shifts comprise two parts, the contact shift and PCS. Contact
shifts are a consequence of the delocalization of unpaired
electron spin density across chemical bonds and thus are only
observed for nuclear spins fairly close to the paramagnetic
center. In contrast to the contact shift, the PCS is a through-
space interaction, which arises from the time-averaged dipolar
interaction between the unpaired electron spins and the nuclear
spin. PCSs are observable over much greater distances and, as
they are independent of bond angles, can be described by fewer
parameters, which pertain to the Δχtensor and the location of
the nuclear spin relative to the Δχtensor. In this way, PCSs
deliver long-range distance and orientation information, which
can readily be interpreted.
Anisotropic magnetic susceptibilities also cause weak align-
ment of the paramagnetic molecule in the external magnetic
field, leading to the observation of RDCs between nuclear spins.
In as far as the molecule tumbles in solution as a single rigid
entity, the alignment affects the entire molecule irrespective of
the location of the paramagnetic center and, therefore, RDCs do
not depend on the distance of the nuclear spins from the
paramagnetic center. Conveniently, the size and orientation of
the alignment tensor are proportional to the size and orientation
of the Δχtensor.
A paramagnetic center with Curie spin generates a magnetic
dipolar field at the site of a nuclear spin. The mathematical
description of the effect of this dipolar field on the magnetic
shielding of the nucleus is closely similar to that of chemical shift
anisotropy (CSA). To highlight this similarity, the term dipolar
shielding anisotropy (DSA) has been coined for this para-
magnetic effect. The similarity extends to cross-correlated
relaxation (CCR) effects between DSA and dipole−dipole
(DD) relaxation, which is manifested in differential relaxation
rates of multiplet components, just like the CSA/DD cross-
correlation effects that form the basis of transverse relaxation
optimized spectroscopy (TROSY).
55,56
DSA/DD cross-corre-
lated relaxation can be used to evaluate the angles between an
internuclear bond and the principal axes of the DSA tensor. As
the magnitude of the DSA tensor depends on the distance of the
nuclear spin from the paramagnetic center, conversion of CCR
effects into structural restraints is more involved than for RDCs.
Nonetheless, these CCR effects are readily observable and can
provide useful distinctions between different pairs of coupled
nuclear spins that otherwise display similar paramagnetic
effects.
57
Adifferent type of CCR effect also occurs between CSA and
DSA relaxation. This CCR effect can significantly affect the
nuclear net relaxation rates, either enhancing or reducing the
relaxation of nuclear magnetization in the paramagnetic state
compared with the corresponding diamagnetic state.
58
In general, paramagnetic centers enhance the relaxation rates
of nuclear spins, which is referred to as PRE. A PRE is the direct
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consequence of dipole−dipole interactions between the para-
magnetic center and a nuclear spin. It decreases with the sixth
power of the distance between them. The large magnitude of the
PRE at short distances and its steep distance dependence render
the PRE a sensitive tool to detect minor states that position the
nucleus close to the unpaired electrons, as may occur in dynamic
proteins carrying a paramagnetic tag. Paramagnetic centers with
slow electronic relaxation (∼109s−1), such as presented by
nitroxide radicals, Mn(II), and Gd(III) ions, generate PREs by
dipole−dipole relaxation, referred to as Solomon−Bloember-
gen−Morgan (SBM) or Solomon relaxation.
59
In contrast, SBM
relaxation is much less efficient for unpaired electrons with fast
electronic relaxation rates (>1011 s−1) and Curie spin relaxation
becomes more prominent,
60,61
especially for long rotational
correlation times of the molecule and at increased magnetic field
strength. In general, the SBM mechanism is the sole
contribution to PREs in isotropic paramagnetic species and
the Curie spin mechanism adds an additional component in
systems characterized by paramagnetic anisotropy, which can
nonetheless become the dominant contribution to PREs.
Many transition metal ions contain unpaired electrons,
leading to paramagnetism. The paramagnetism of 3d block
and 4f block ions has been studied most thoroughly.
29,62
Mn(II)
ions have a long electronic relaxation time, causing no PCSs but
strong PREs. High-spin Co(II) features a Curie spin and can
generate large PCSs with weak PREs, compared with other 3d
block ions. In aqueous solution, iron is stable in the oxidation
states +2 and +3. Depending on its ligands, Fe(II) can be
paramagnetic or diamagnetic. Similarly, the paramagnetism of
Fe(III) can be either large (high spin) or small (low spin),
depending on the coordination environment. The 4f block
elements are referred to as lanthanoids,
63
with the general
symbol Ln. Unlike 3d block ions, lanthanoids are seldom
involved in biological processes, with the first examples of
lanthanoenzymes discovered only in 2011 in methanotrophic
bacteria.
64
Lutetium(III) and lanthanum(III) have no unpaired
electrons, while all other Ln(III) ions of the series are
paramagnetic. Gd(III) ions are unique for featuring a strong
isotropic magnetic susceptibility, which is generated by seven
unpaired electrons. Gd(III) ions yield exceptionally strong PREs
without generating PCSs. Other Ln(III) ions have Curie spins of
different magnitude, causing both PCS and PRE effects. Stable
organic radicals such as nitroxides or the trityl radical can also be
used to create a paramagnetic center. They do not cause PCSs,
but yield sizable PREs.
Quantification of the paramagnetic effects is commonly
achieved by comparison with a diamagnetic species. Therefore,
the appropriate choice of a diamagnetic reference as similar to
the paramagnetic sample as possible is very important.
Lanthanoids are chemically similar to each other, so that a
diamagnetic reference can be obtained easily by substitution of
the paramagnetic ion with Lu(III) or La(III). Furthermore, the
ionic radius of Y(III) is practically the same as that of Ho(III),
making it an excellent diamagnetic reference for the heavy
lanthanoid ions ranging from Tb(III) to Yb(III). For this reason,
the present article makes no formal distinction between Ln(III)
and Y(III) ions. In the case of nitroxide radicals, the diamagnetic
reference is usually obtained by chemical reduction to the
corresponding hydroxylamine, which is readily achieved with
ascorbic acid. Alternatively, diamagnetic control probes (such as
hydroxylamine derivatives) can be used that are chemically
similar to the nitroxide probes.
65
As the properties of 3d block
ions vary more than those of Ln(III) ions, it is more difficult to
identify suitable diamagnetic references. Zn(II) is commonly
used as a diamagnetic reference for Co(II) and Mn(II) because
charge and size are similar, and Ga(III) has been successfully
used as a diamagnetic reference for high-spin Fe(III).
66,67
1.3. Quantitative Description of Paramagnetic Effects in
NMR Spectroscopy
Mathematical descriptions of the different paramagnetic effects
are well established and enable their quantitative interpretation.
Hyperfine shifts, such as PCSs, are reported as the change in
chemical shift (measured in ppm) caused by the presence of a
paramagnetic center. RDCs are measured in hertz, reporting on
the change in multiplet splitting observed due to weak
paramagnetic alignment of the molecules with the magnetic
field. PREs and cross-correlated relaxation effects are measured
in s−1, presenting the paramagnetic enhancement in longitudinal
(R1) or transverse (R2) relaxation rates of nuclear spins over a
corresponding diamagnetic reference, with R1and R2being the
inverse of the T1and T2relaxation times. The basic equations
governing the various paramagnetic effects have been treated in
numerous review articles and books.
7−9,11,13,19,68
In the
following, we present a summary to highlight the salient features
together with correction terms that may become relevant in
special circumstances.
Figure 1. Schematic representation of the paramagnetic effects of PCS, RDC, PRE, and CCR, respectively, where CCR refers to cross-correlation
between DSA and dipole−dipole relaxation. The top panel depicts the geometric dependencies relative to the frame of a χtensor. The bottom panel
illustrates the effects in the NMR spectra of paramagnetic (red) versus diamagnetic (black) samples.
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D

1.3.1. Pseudocontact Shift (PCS). The PCS of a nuclear
spin can be described by eq 1
7
δπχθ
χθϕ
Δ= Δ −
+Δ
Ä
Ç
Å
Å
Å
Å
Å
Å
Å
ÅÉ
Ö
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
r
1
12 (3 cos 1)
3
2(sin cos 2 )
PCS
3ax
2
rh
2
(1)
χχ
χχ
χχχ
Δ
=− +Δ=−
2and
z
xy
x
y
ax rh (2)
where ris the distance between the paramagnetic center and the
nuclear spin, and the angles θand ϕare the polar angles
describing the location of the nucleus with respect to the
principal axes of the χtensor. The axial component of the
magnetic susceptibility anisotropy, Δχax, and the rhombic
component Δχrh are defined by eq 2, where χx,χy, and χz
denote the values of magnetic susceptibility along the respective
principal axes of the χtensor (Figure 1A). Being defined as the
anisotropic component of the χtensor, the Δχtensor is spanned
by principal axes that are aligned with those of the χtensor. Use
of eq 1 for calculating the PCS of a nuclear spin in a given
molecular structure requires the prior knowledge of the location
of the paramagnetic center and the orientation of the Δχtensor
relative to the molecule. The orientation of the Δχtensor is
commonly described by three Euler angles (α,β, and γ) and, in
the case of proteins, reported relative to coordinates deposited in
the protein data bank (PDB). Together with the Δχax and Δχrh
values, eight parameters are thus required to define the Δχ
tensor relative to a set of atomic coordinates. They can be
obtained by using the experimentally measured PCS data of at
least eight different nuclear spins to fit the Δχtensor to the
molecular structure. A number of software packages
69−73
are
available to perform this fit. In practice, good fits require at least
three times more PCS data than the minimum of eight, in which
case it is also possible to obtain a measure of the uncertainties of
the Δχtensor parameters by Monte Carlo random variation of
the data input.
To fitΔχtensors to molecular structures and calculate
correction terms arising from cross-correlation effects and
molecular alignment, it is most convenient to use a matrix
representation of the χtensor. The isotropic component of the χ
tensor is given by
χμμ
=[+]SS
kT
g(1)
3
e
iso
0
2
B
2
B(3)
where μ0is the induction constant and geand Sare the electronic
gfactor and spin, respectively; their values are replaced by gJand
Jfor lanthanoid ions.
74
μBis the Bohr magneton; kBdenotes the
Boltzmann constant, and Tis the absolute temperature.
Including the anisotropic components of the χtensor, a concise
mathematical description of the dipolar shift tensor σat a given
position rand distance rfrom the paramagnetic center can be
written as

σπχ
π
χχχ
χχχ
χχχ
=⊗−·
=
−
−
−
·
Ä
Ç
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
É
Ö
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ä
Ç
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
É
Ö
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ä
Ç
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
É
Ö
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
rr
r
rr
xr xy xz
xy y r yz
xz yz z r
1
43
1
4
(3 ) 3 3
3(3)3
33(3)
T
xx xy xz
xy yy yz
xz yz zz
53
5
3
22
22
22
(4)
where

3denotes the 3 ×3 identity matrix, ⊗denotes the
Kronecker product, and x,y, and zthe coordinates of the nucleus
relative to the origin of the χtensor.
7,73
(Note that the dipolar
shift tensor is the negative of the dipolar shielding tensor.) The
full χtensor including its isotropic component is needed to
calculate PREs, but the Δχtensor suffices to calculate PCSs. The
Δχtensor in matrix representation is the traceless part of the χ
tensor (i.e., the same as the χtensor, except that each diagonal
element is reduced by their average). In the representation of eq
4, the PCS is given by the trace of the shielding tensor
δσ
Δ
=[]Tr
1
3
PCS
(5)
which can also be expressed in terms of Δχtensor elements
δπ
Δ
=[− − ]·
Δ
Δ
Δ
Δ
Δ
Ä
Ç
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
É
Ö
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
rx z y z xy xz yz
x
x
x
x
x
1
4,,2,2,2
xx
yy
xy
xz
yz
5
PCS 2 2 2 2
(6)
Eq 6 illustrates how separating the position coordinates of the
PCS equation (eq 5) from the Δχtensor leads to a linear form of
the equation with 5 unique elements in a column vector defining
the tensor. Equation 6 thus allows Δχtensor fits for a fixed
position by singular value decomposition. Combined with the
three coordinates of the metal center relative to the coordinates
of the molecule, a Δχtensor fit thus requires determining eight
parameters.
1.3.2. Residual Dipolar Coupling (RDC). In isotropic
solutions, internuclear dipolar couplings average to zero due to
fast molecular tumbling, but RDCs re-emerge upon partial
alignment of the molecules in a magnetic field. RDCs are most
easily measured for one-bond scalar couplings, where they
manifest in altered distance (measured in Hz) between well-
separated multiplet components.
27
If the molecular alignment is
caused by a paramagnetic center with anisotropic magnetic
susceptibility, the alignment tensor Ais directly proportional to
the Δχtensor, with the same axes directions
7
χ
μ
=Δ
A
B
kT15
0
2
0B(7)
where B0is the magnetic field strength. The residual dipolar
coupling between two nuclear spins Iand Kis described by
7,75
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E

γγ
πχ
χ
=− Δ Θ−
+Δ Θ Φ
Ä
Ç
Å
Å
Å
Å
Å
Å
Å
ÅÉ
Ö
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
DhB
rkT240 (3 cos 1)
3
2sin cos 2
IK
0
2
IK
3IK
3Bax
2
rh
2
(8)
where γIand γKdenote the gyromagnetic ratios of the nuclear
spins Iand K, respectively, rIK is the internuclear distance, h
Planck’s constant, and the angles Θand Φdetermine the
orientation of the I−Kvector relative to the Δχtensor (Figure
1B). The RDC, thus, yields information about the orientation of
the internuclear vector in the frame of the Δχtensor
independent of the distance of the nuclei from the paramagnetic
center. RDCs allow fitting the alignment tensor to a protein
structure and, once the tensor frame has been determined, the
RDCs can be used to establish bond vector orientations within a
protein structure. As reorientational motions of a bond vector
reduce the RDC by averaging over different orientations, RDC
measurements can be used to study protein dynamics.
27,76
1.3.3. Residual anisotropic chemical shift (RACS). The
weak molecular alignment in the magnetic field caused by
anisotropic magnetic susceptibilities not only produces RDCs,
but also changes the chemical shifts of nuclear spins that feature
significant CSA tensor anisotropies by rendering the averaging
over different molecular orientations incomplete. The change in
chemical shift caused by such RACS effects poses a limit on the
accuracy with which PCSs can be measured. If the structure of
the molecule is known, the CSA tensors associated with
individual nuclei can be taken into account by estimating a
RACS correction for each PCS measured. The corrections can
be significant at high magnetic field strength,
77
as the degree of
molecular alignment depends on the square of the magnetic field
(eq 7).
1.3.4. Residual Anisotropic Dipolar Shift (RADS).
Residual anisotropic dipolar shifts present another effect by
which experimentally measured PCS data may have to be
corrected to obtain the pure PCS described by eq 1. Like the
RACS effect, the RADS effect arises from weak molecular
alignment in the magnetic field, which leads to incomplete
averaging of anisotropic chemical shifts. Paramagnetic centers
with a Curie spin create a dipolar shielding tensor at the site of
the nuclear spin, which is generally anisotropic and similar to a
CSA tensor. As molecular alignment results in nonuniform
averaging of the dipolar shielding, the PCS produced by the
effective DSA tensor is no longer fully represented by eq 1.
Fortunately, RADS corrections can be calculated and,
importantly, are barely measurable in practice.
7
1.3.5. Saturation Effect. Finally, saturation of magnetic
moments at high magnetic field strength renders the Δχtensor
and, hence PCSs, field dependent. This effect is small even at
18.8 T (800 MHz for 1H NMR) but may become more
important at much higher field strengths.
78
1.3.6. Paramagnetic Relaxation Enhancement (PRE).
Spin−spin interactions are an important source of nuclear
relaxation. Due to the large magnetic moment of unpaired
electrons, electron−nuclear spin interactions easily provide a
dominant source of nuclear relaxation. The SBM relaxation
mechanism
59,79,80
applies to all paramagnetic centers. For
paramagnetic centers with unpaired electrons that relax rapidly
within the rotational correlation time of the molecule, the Curie
spin mechanism
60,61
provides an additional and often dominant
source of PREs. SBM relaxation describes the effect from
dipole−dipole coupling between electron and nuclear spins, as it
drives nuclear relaxation by the variation in dipolar fields due to
molecular reorientation and changes in the electronic spin state
due to longitudinal electron relaxation. The paramagnetic
enhancements in longitudinal (R1
SBM) and transverse (R2
SBM)
relaxation rates can be described by
μ
π
γμ τ
ωτ
τ
ωτ
=+
+
++
i
k
j
j
jy
{
z
z
z
i
k
j
j
j
j
j
y
{
z
z
z
z
z
R
SS
r
2
15 4
g(1)3
1
7
1
1
SBM 02I
2
e
2
B
2
IS
6
c
I2c
2
c
S
2c
2(9)
μ
π
γμ ττ
ωτ
τ
ωτ
=+++
++
i
k
j
j
jy
{
z
z
z
i
k
j
j
j
j
j
y
{
z
z
z
z
z
R
SS
r
1
15 4
g(1)
43
1
13
1
2
SBM 02I
2
e
2
B
2
IS
6cc
I2c
2
c
S
2c
2(10)
τ
ττ=+
−−−
c1r1s1(11)
where rIS is the distance between nuclear and electron spin and
ωIand ωSare the nuclear and electron Larmor frequencies,
respectively. Electronic spin flips are accounted for by the
effective correlation time τc, which is composed of the rotational
correlation time τrand the electronic lifetime τsas shown by eq
11. Terms in eqs 9 and 10 that depend on the electron
frequencies can usually be neglected unless the lifetime of the
electronic spin states is sufficiently short to approach the inverse
of the electron precession frequency. The steep distance
dependence of the PRE makes it very sensitive to changes in
rIS, which has been exploited to detect little populated
conformations and protein−ligand complexes in solution.
81−83
Curie spin relaxation likewise results from dipole−dipole
coupling between electron and nuclear spins, but it arises from
the dipolar field created by the average electronic polarization
due to higher populations of lower-energy as opposed to higher-
energy electronic spin states.
84
Curie spin relaxation applies to
systems in which electron relaxation occurs in a time short
compared with the rotational correlation time of the molecule.
Nuclear relaxation is insensitive to magnetic fields fluctuating at
rates that are orders of magnitude faster than the Larmor
frequency, such as the very fast electronic spin flips associated
with Curie spins. Curie spin relaxation is driven by rotational
tumbling, with the rotational correlation time τrcharacterizing
the time-dependent modulation. Because of a quadratic
dependence on the nuclear Larmor frequency, Curie spin
relaxation can outweigh SBM relaxation for high-molecular
weight systems in a strong magnetic field
85,86
(note that the
calculations of Table 2 in ref 85 were performed for τr=10ns
instead of τr= 1 ns as stated in the original) while decreasing
noticeably with increasing temperature.
9
A quantitative
description of PREs by Curie spin relaxation is given by eqs
12 and 13 for longitudinal (R1
Curie) and transverse (R2
Curie)
relaxation rates.
μ
π
ωμ τ
ωτ
=+
+
i
k
j
j
jy
{
z
z
z
i
k
j
j
j
j
j
y
{
z
z
z
z
z
R
gSS
kT r
2
45 4
(1)
()
3
1
1
Curie 02I2
e
4
B
42 2
B2IS
6
r
I2r2(12)
μ
π
ωμ ττ
ωτ
=+++
i
k
j
j
jy
{
z
z
z
i
k
j
j
j
j
j
y
{
z
z
z
z
z
R
gSS
kT r
1
45 4
(1)
() 43
1
2
Curie 02I2
e
4
B
42 2
B2IS
6rr
I2r2
(13)
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F

Eqs 9−13 describe the situation of isotropic magnetic
moments associated with the paramagnetic center. In general,
however, magnetic moments often are anisotropic. At the
atomic level, such anisotropies usually arise when the magnetic
moment of a paramagnetic metal ion depends not only on the
electronic spin but also on spin−orbit couplings and therefore
on the ligand field. This situation is particularly prominent for
paramagnetic lanthanoid ions (except Gd(III)) and therefore
their time-averaged magnetic moment is best described by an
anisotropic molecular magnetic susceptibility tensor. For
complexes with Sm(III) and Eu(III) ions, the situation is
further complicated by a significant population of low-lying
excited electronic states with different paramagnetic character-
istics, which is manifested by a temperature dependence that is
less steep than predicted by eqs 12 and 13.
74
An extension to the SBM theory has recently been described,
which accounts for anisotropic dipolar spectral density in terms
of a spectral power density tensor.
87
As this tensor usually
cannot be derived theoretically, however, this extended theory
depends on a greater number of parameters to be fitted to the
experimental data. The anisotropic SBM theory is relevant for
SBM relaxation generated by lanthanoid ions with anisotropic χ
tensors. For these ions, however, Curie spin relaxation often
exceeds SBM relaxation, making it difficult to detect the
anisotropy effects in the SBM relaxation contribution.
The impact of anisotropic χtensors on Curie spin relaxation
can be calculated.
61
Using the matrix formalism of eq 4, the R1
Curie
and R2
Curie rates can be written
σσ σσ σσ
Λ
=− +− +−()()()
xy yx xz zx yz zy
222 2
(14)
σσσσσσσσσ
σσ σσ σσ
Δ= + + − − −
+[ + + + + +
]
3
4()()()
xx yy zz xx yy xx zz yy zz
xy yx xz zx yz zy
22 2 2
222
(15)
ωτ
τω ωτ
ωτ
=Λ ++Δ +
Ä
Ç
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
É
Ö
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ä
Ç
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
É
Ö
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
R
1
219
2
15 1
1
Curie 2 2 r
r22
22 r
2r2
(16)
ωτ
τω ωτ τ
ωτ
=Λ ++Δ +
Ä
Ç
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
É
Ö
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ä
Ç
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
É
Ö
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
R
1
419
1
45 43
1
2
Curie 2 2 r
r22
22 rr
2r2
(17)
where ωrefers to the Larmor frequency of the nuclear spin. The
anisotropy effects tend to be small and not usually apparent in
PRE measurements. Limited evidence has been found in cross-
correlation effects.
88
1.3.7. Cross-correlated Relaxation (CCR). 1.3.7.1. DSA/
CSA Cross-correlation. For systems with Curie spins, the
dipolar field emanating from the paramagnetic center
contributes a variable magnetic field at the site of the nuclear
spin, which drives nuclear relaxation in a way akin to chemical
shift anisotropy. This analogy is highlighted by describing Curie
spin relaxation as DSA relaxation. The matrix formalism of eq 4
automatically includes the effect from cross-correlation between
DSA and CSA relaxation, as the effective shielding tensor at the
site of the nuclear spin, σeff, is the sum of the DSA and CSA
tensors. Experimentally, the PRE including cross-correlated
relaxation is obtained as usual by subtracting the relaxation rate
measured for the diamagnetic reference, R(σCSA), from the
relaxation rate in the paramagnetic state, R(σeff), where both
terms can be calculated using eqs 14−17.
σσ=−
·
R
RR() ( )
Curie CSA eff CSA (18)
For nuclei for which CSA relaxation is the main relaxation
mechanism in the diamagnetic state, the contribution by the
DSA/CSA cross-correlation effect can be dominant. In this case,
it is possible that the σeff tensor becomes more isotropic than the
CSA tensor because of fortuitous compensation by the DSA
tensor, which is manifested by lesser relaxation rates in the
paramagnetic than in the diamagnetic state. This was predicted
in 2004
85
and experimentally observed for the first time in
2016.
58
The effect was demonstrated with negative PREs of 15N
nuclei.
1.3.7.2. DSA/DD Cross-correlation. Just as CSA/DD cross-
correlation effects give rise to differential broadening effects of
individual multiplet components (which has been exploited, for
example, in TROSY spectra), DSA/DD cross-correlation effects
also create differential broadening effects of multiplet
components.
89
Using the matrix representation of eq 4, the
cross-correlation effect can be calculated for the example of a 1H
spin that is bonded to a 15N nucleus by

σμ
πγ=ℏ
⊗−
Ä
Ç
Å
Å
Å
Å
Å
Å
Å
Å
Å
Å
É
Ö
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
B
rr
rr
1
4I3
T
N
0
0
N
HN HN
HN
5
3
HN
3(19)
σσσ=+
↑
N
(20)
σσσ=−
↓
N
(21)
σσ=−
·↑↓
R
RR() ()
Curie DD Curie Curie
(22)
where σNdenotes the shift tensor at the site of the 1H spin, I, that
originates from the dipolar field of the 15N spin and adds either
positively or negatively to the full shielding tensor of the 1H spin,
depending on the spin state of the 15N nucleus (as described by
σ↑and σ↓). With the complete shift tensors at hand, the
differential line broadening observed between the doublet
components of the 1H spin, RCurie·DD, is readily calculated by
using eqs 4,14−17, and 19−22.
CSA/DD cross-correlation effects only appear in NMR
spectra recorded without broadband decoupling. The value of
the associated structural information has been demonstrated in a
3D structure of calculation of cytochrome c′from Rhodobacter
capsulatus using paramagnetic restraints only.
90
1.3.7.3. Accuracy of ΔχTensor Determination. The matrix
representation of the Δχtensor of eq 6 facilitates the fitting of
Δχtensors to the atomic coordinates of the macromolecular
structure by a linear least-squares fit, using experimentally
observed PCSs. The quality of the fit is commonly described by a
quality factor Q, where a low value indicates a good fit:
=
∑∑
[−]
∑[∑[]]
Ä
Ç
Å
Å
Å
Å
Å
Å
É
Ö
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Q
aa
a
()
()
im
iexp m, i
cal 2
im
iexp 2
(23)
where aexp and acal are the experimental and calculated PCSs
values, respectively, the index mindicates ensemble averaging of
spins that are common between different models of the
molecular structure, and the index iis for summation over all
spins of the molecule. Alternative Qfactors have been
proposed.
91,92
The Qfactor proposed by Bashir et al.
91
uses
sums of experimental and calculated values in the denominator
of eq 23 and, therefore, tends to be 2 times smaller. This
definition has the advantage that it does not bias for calculated
values that are smaller over those that are larger than the
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experimental values, which matters in the case of poor fits with
large differences between observed and calculated data, as can be
the case with PREs. For example, for (aexp,acal) of (10, 20) Q=1,
but for (20, 10), Q= 0.5. The adjusted Qfactor yields 0.33 in
both cases. Importantly, Qfactors are meaningful only, if the
number of fitted data greatly exceeds the number of variables, as
very small values in the denominator can render its calculation
unstable. Therefore, fitting algorithms usually attempt to
minimize the root-mean-square deviation (RMSD) between
calculated and experimental values rather than Qfactor.
In the case of paramagnetic metal probes attached to the
target molecule via a long and flexible linker, the range of
positions assumed by the metal ion relative to the target calls for
an equal range of Δχtensors to be fitted. Fitting multiple tensors,
however, usually is not possible as each Δχtensor determination
requires at least eight PCSs to fit the tensor parameters and
accurate fits require significantly more PCSs. The attempt to fit
more than a single Δχtensor to the limited data available would
easily lead to problems with overfitting. The convention to fit
single effective Δχtensors has two consequences. First, the
location of the paramagnetic center obtained by the fit does not
correspond to its real position. In fact, it is quite possible that,
due to temporary close proximity of the paramagnetic metal ion,
the PCSs observed for a range of nuclear spins near the
attachment site of the paramagnetic tag are larger than expected
for a single immobile metal position. In this case, fitting of the
data by a single effective Δχtensor tends to increase the Δχ
tensor and indicate a metal position that is further away from the
surface of the target molecule than expected. This does not
invalidate the use of effective Δχtensors to back-calculate PCSs,
but the predictive value of PCSs decreases with increasing
distance from the nuclear spins, whose PCS data were used to fit
the Δχtensor.
93
Notably, exceptionally good correlations
between experimental and back-calculated PCSs can be
obtained even with tags attached via flexible linkers.
94
The difficulty to determine accurate Δχtensors for flexible
probes makes it difficult to assign the magnitudes of reported Δχ
tensors to intrinsic probe characteristics. When a large number
of PCSs has been used for the Δχtensor fit, fits performed for
rigid probes tend to produce low Qfactors. An alternative way of
assessing the flexibility of a paramagnetic probe is to compare
the alignment tensor obtained by fitting RDCs with the Δχ
tensor obtained by fitting PCSs, as both should be proportional
to each other (eq 7). In practice, however, the alignment tensor
is almost always smaller than that derived from PCSs because (i)
RDCs are very sensitive to the accuracy of the molecular
coordinates and (ii) molecules are not rigid and the orientations
of the internuclear vectors determining the RDCs are averaged
due to molecular dynamics as reflected by order parameters
below one.
1.4. Paramagnetic NMR of Biomolecules in the Solid State
Solid-state NMR spectroscopy of biological macromolecules
such as microcrystalline, fibrillar and membrane proteins is a
growing area of interest largely driven by recent advancements in
magic angle spinning (MAS) technology.
95,96
Rotation speeds
beyond 100 kHz have been shown to allow the acquisition of 1H-
detected multidimensional NMR spectra with good resolution
and sensitivity without the need of protein perdeuteration.
97,98
The first paramagnetic macromolecules to be investigated by
solid-state NMR were metalloproteins, such as Co(II)
substituted matrix metalloproteinase-12, for which PCSs were
measured to characterize protein structure.
99−101
Co(II)-
substituted superoxide dismutase was also investigated by
PCSs and PREs with 1H detected ultrafast MAS to determine
molecular structure.
102,103
Paramagnetic tags attached to
biomolecules offer similarly useful long-range structural
information in the solid state as in solution, but there are
differences in the paramagnetic effects.
53,52
1.4.1. Paramagnetic NMR Effects in Static and
Rotating Solids. An unordered paramagnetic compound in
the solid state features molecular orientations distributed
uniformly. As the dipolar shielding tensor arising from the
paramagnetic center (see eq 4) renders the chemical shift for the
nuclear spin dependent on the orientation of the molecule, the
resulting powder pattern in the NMR spectrum reflects the
principal axes of the σtensor.
For the case of rotating solids, MAS of the paramagnetic
species achieves coherent averaging of the σtensor. At low
spinning rates, this tends to split the powder pattern into
spinning sidebands.
104
When the spinning rate is greater than
the dipolar shielding term, however, only the isotropic
component of the σtensor, which includes the PCSs, remains
observable in the NMR spectrum and can be described by eq
1.
105
While incoherent averaging of the σtensor in solution NMR
underpins the Curie spin relaxation mechanism as described in
eqs 12−13, the coherent averaging by MAS effectively removes
this pathway as a source of PREs in solid-state NMR.
106
Therefore, PREs of nuclear spins are unaffected by the Curie
mechanism (except for nuclei in close proximity to the
paramagnetic center, where the σtensor term becomes larger
than the MAS rate) and SBM relaxation (eqs 9−10) presents the
dominant source of PREs in solids. SBM relaxation is governed
by the incoherent electronic correlation time T1e and
independent of the MAS rate.
59
2. SOURCES OF PARAMAGNETISM
This section presents a brief overview of the most frequently
used paramagnetic metal ions and nitroxide radicals.
2.1. 3d Block Transition Metal Ions
Transition metals in the 3d block of the periodic table can be in
various oxidation states with different numbers of unpaired
electrons occupying the d-orbitals.
107
Therefore, these cations
are great candidates for generating various paramagnetic effects.
They are frequently found in proteins. It is estimated that more
than 25% of the known proteins contain one or more transition
metal ions.
108
Paramagnetic NMR has long been applied to
investigate the metal binding sites of metalloproteins.
109−111
The first 3D structure determination of a metalloprotein was
performed on a heme protein containing a low-spin Fe(III)
ion.
112
Proteins containing many other paramagnetic transition
metal ions have been studied, with iron, cobalt, manganese,
copper, and nickel ions figuring most frequently. These ions are
discussed hereafter.
Transition metal ion complexes of 3d block elements are
stable with 4−6 electron donor sites. Depending on the number
of coordination sites, transition metal ions feature mainly three
types of coordination geometries, which are tetrahedral, square
pyramidal, and octahedral (Figure 2). The magnetic properties
of 3d block complexes can be strongly affected by the ligands, the
environments, and the coordination geometries. Some general
properties are shown in Table 1. The unpaired electrons usually
are partially delocalized to the ligand orbitals, which affects the
magnetic properties. Iron is a good example. Fe(III) and Fe(II)
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are the most stable and commonly found oxidation states of iron.
Both of them can be in paramagnetic high-spin states and
Fe(III) is also paramagnetic in the low-spin state. With weak
axial ligands, Fe(III) is in a high-spin state, whereas Fe(III)
assumes a low-spin state with strong ligands. High-spin Fe(III)
contains five unpaired electrons, and its electronic relaxation
time can vary from 10−9to 10−11 s.
113,114
Therefore, it can
generate sizable PCSs and PREs, depending on the ligands. The
magnetic properties of low-spin state Fe(III) are quite different
due to its short electronic relaxation time, which is below 10−11
s.
113,114
As a result, the paramagnetism of low-spin Fe(III) is
most prominently manifested in PCSs. Fe(II) has six electrons
occupying d-orbitals, so when these electrons are paired, Fe(II)
is diamagnetic. Its high-spin state has up to four unpaired
electrons with a short electronic relaxation time (10−12 s), which
causes PCSs.
107
Proteins containing iron ions have been well
studied by paramagnetic NMR, for example cytochrome c,
rubredoxin, and hemoglobin.
62
The electronic configuration of Co(II) is 3d7, which can be in
a high-spin state containing three unpaired electrons or in a low-
spin state containing a single unpaired electron. The electronic
relaxation times of low-spin Co(II) are longer (10−9−10−10 s)
than for the high-spin state (∼10−12 s), thus low-spin Co(II)
causes strong PREs and small PCSs.
107
In contrast, high-spin
Co(II) can generate the largest PCSs among 3d block ions
combined with weak PREs. Mn(II) harbors one of its five
unpaired electrons in each of the d-orbitals and has a long
electronic relaxation time, causing the strongest PREs of all 3d
block ions. Cu(II) is the most stable ionic state of copper. Three
types of Cu(II) ions are found in proteins, which are
distinguished by the Cu(II) ion coordination.
15,62
In type-I
copper(II) proteins, the ion is coordinated with trigonal (or
distorted tetrahedral) geometry, involving at least one sulfur
atom of cysteine and two nitrogens of two histidine residues.
115
In type-II copper(II) proteins, the copper ion is coordinated by
nitrogen and oxygen atoms with tetrahedral geometry. In the
third type, two Cu(II) ions are antiferromagnetically coupled,
decreasing the net magnetic susceptibility and electron
relaxation time.
116
Consequently, various paramagnetic effects
were observed for Cu(II). Cu(II) contains one unpaired
electron and generates small PCSs, but the PREs are strong,
because of its relatively long electronic relaxation time (10−8−
10−9s). Therefore, Cu(II) is outstanding for PREs and it is
widely used in EPR spectroscopy.
107,117,118
The divalent ion of
nickel has two unpaired electrons, generating small PCSs with
sizable PREs.
107
2.2. Lanthanoid Ions
Lanthanoid ions were introduced into NMR studies of proteins
decades ago, as agents for chemical shift changes and line
broadening.
119,120
The chemical properties of all Ln(III) ions
are similar because the 4f-orbitals are shielded by 5s and 5p
subshells, and their unpaired electrons do not participate
substantially in different coordinating interactions with ligands.
Ligands that bind Ln(III) ions thus have similar affinities for all
lanthanoid ions. An overview of the paramagnetic properties of
paramagnetic lanthanoid ions (excluding promethium, which is
an unstable radioactive element) is presented in Table 2.
Gd(III) is unique because its seven unpaired electrons are
distributed equally among the f-orbitals, producing a magnetic
dipole moment that is independent of molecular orientation in
Figure 2. Schematic diagrams of 3d block ions coordination
geometries.
Table 1. Magnetic Properties of 3d Block and 4f Block Ions
ion conf.
a
J
b
τs
c
range
d
PCSs
e
PREs
e
RDCs
e
Fe(III) (HS) [Ar]3d55/2 10−9−10−13 20 + ++
Fe(III) (LS) [Ar]3d51/2 10−11−10−13 15 ++ +
Mn(II) [Ar]3d55/2 10−825 ++
Fe(II) (HS) [Ar]3d6210
−12 15 + +
Co(II) (HS) [Ar]3d73/2 10−12 25 ++ +
Ni(II) [Ar]3d8110
−10−10−12 15 + +
Cu(II) [Ar]3d91/2 10−8−10−920 + ++
Ce(III) [Xe]4f15/2 10−13 10 + +
Pr(III) [Xe]4f2410
−13 20 + +
Nd(III) [Xe]4f39/2 10−13 10 + +
Sm(III) [Xe]4f55/2 10−13 7+ +
Eu(III) [Xe]4f6010
−13 15 + +
Gd(III) [Xe]4f77/2 10−825 +++
Tb(III) [Xe]4f8610
−13 45 ++++ + +++
Dy(III) [Xe]4f915/2 10−13 45 ++++ + +++
Ho(III) [Xe]4f10 810
−13 35 +++ + ++
Er(III) [Xe]4f11 15/2 10−13 30 ++ + +
Tm(III) [Xe]4f12 610
−12−10−13 50 +++ ++ ++
Yb(III) [Xe]4f13 7/2 10−13 25 ++ + +
Nitr.
f
1/2 10−715 ++
a
Electronic configuration of the ions.
b
Total angular momentum quantum number.
c
Electronic relaxation time (in seconds) reported in refs 29,86,
735, and 736.
d
Range of paramagnetic effect (in Å) reported in ref 29,107, and 86.
e
+ indicates the intensity of the effect based on refs 29,107, and
86. No entry indicates not applicable or small effect.
f
Nitr., nitroxide.
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I

an external magnetic field. As its electronic relaxation time is
long (>10−8s) at field strengths >3 T,
29,86
Gd(III) generates
large PREs and enjoys increasing popularity in EPR as spin
label.
121−123
Although the remaining paramagnetic lanthanoid
ions generate anisotropic χtensors and produce all the
paramagnetic effects discussed above, it has recently been
shown that PRE measurements with Er(III), which generates
large PREs associated with relatively small PCSs, can yield more
accurate distance measurements than commonly used para-
magnetic agents with long electronic relaxation times.
124
In
general, Tb(III) and Dy(III) generate the largest Δχtensors
and, hence, cause the largest PCSs, while Tm(III) and Ho(III)
produce medium-sized PCSs. Sizeable PCSs can also be
observed with Yb(III) and Er(III). This general ordering,
which follows theoretical expectations based on gJfactor and J
quantum number
74
and has been experimentally verified for
calbindin D9k with one of the native calcium ions replaced by a
Ln(III) ion,
125
is not always maintained as, for reasons not
understood at present, some complexes of Tm(III) have been
found to produce larger Δχtensors than the same complexes
with Dy(III) or Tb(III).
126−129
Other lanthanoid ions are much
less frequently used for paramagnetic NMR of proteins because
their Δχtensors are smaller.
86
2.3. Nitroxide Radicals
Nitroxide probes are organic molecules with five- or six-
membered heterocyclic rings and a radical that is protected
against homodimerization by bulky chemical groups (Figure 3).
Because of their relatively small size combined with a long
electronic relaxation time of a single unpaired electron and its
reasonably well-defined localization, nitroxides are the most
frequently used paramagnetic compounds in NMR to generate
PRE distance restraints up to 20−25 Å.
130
The stability of the
radical depends on the size of the ring and the substituents in the
αposition. In general, nitroxides in a five-membered ring are
chemically more stable than in six-membered rings and bulkier
groups in the αposition promote stability by improved steric
shielding. MTSL ((1-oxyl-2,2,5,5-tetramethyl-D-pyrroline-3-
methyl)-methanethiosulfonate) is commercially available and
the most popular nitroxide for protein labeling (Figure 3).
3. GENERAL OVERVIEW OF NATURAL AND
CHEMICALLY GENERATED PARAMAGNETIC
CENTERS
Paramagnetism of biomolecules can exist naturally or be
introduced artificially by different strategies. Metalloproteins
containing paramagnetic ions can readily be studied by NMR
techniques tailored to paramagnetic samples. In some instances
of diamagnetic metalloproteins, a diamagnetic metal ion can be
replaced by a paramagnetic ion. Not all of the diamagnetic
metalloproteins are suitable for metal ion substitution, however,
and this approach does not work for nonmetalloproteins.
Consequently, to gain access to the long-range structural
restraints associated with paramagnetism, various methods have
been devised to introduce paramagnetic centers. Two types of
approaches can be distinguished. Paramagnetic centers can be
added to the solution, resulting in solvent PREs. In this case, the
PREs of protein nuclei are caused by nonspecific interactions
with the paramagnetic relaxation agent in the solution. Such
agents are designed to tumble freely and independently of the
protein molecules, so that any anisotropy of the paramagnetic
center will average to zero, thus yielding only PREs and
Table 2. Paramagnetic Properties of Paramagnetic and Nonradioactive Lanthanoid Ions
a
a
The radii of the yellow spheres correspond to the distance, where paramagnetic relaxation enhancement is predicted to broaden a 1H NMR signal
by 80 Hz on a 800 MHz NMR spectrometer, assuming a protein with a rotational correlation time of 15 ns and a temperature of 25 °C, calculated
using eqs 10 and 13. The isotropic component of the χtensors (χiso,in10
−32 m3) were calculated using eq 3 with values for Sand getaken from ref
74. The Δχtensors reported for calbindin D9k
125
are represented by isosurfaces of the PCSs drawn at ±5 ppm. The values of the Δχtensors are
given in the unique tensor representation,
69
with their relative orientations corresponding to those reported for calbindin D9k. The electronic
longitudinal relaxation times were calculated for a magnetic field strength of 0.5 T following ref 737. They are not strongly field dependent.
738
The
T1e time of gadolinium increases with the rotational correlation time and the square of the magnetic field strength.
739
The scale bar at the left
indicates a distance of 20 Å.
Figure 3. General structure of nitroxides. R represents an alkyl group.
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J

suppressing PCSs or RDCs. The probes for generating solvent-
PREs are usually chemically synthesized.
35,36
Alternatively, a paramagnetic center can be attached
covalently at a specific site on the protein. The two main
methods of the covalent approach are the introduction of a
genetically encoded metal binding site in the target protein and
the chemical attachment of a paramagnetic center to the protein.
Several principles need to be followed in the design of a suitable
protein paramagnetic center. (i) The structure and properties of
the target protein should be maintained. A highly charged or
hydrophobic paramagnetic center as well as improper attach-
ment sites can cause unfolding of the target protein and result in
precipitation. Thus, small probes with low charge and high water
solubility are preferred. (ii) To exploit the effects associated with
paramagnetic metal ions featuring anisotropic χtensors, the
metal ion needs to be attached rigidly to the target protein, as the
anisotropic effects decrease dramatically with the mobility of a
paramagnetic center relative to the protein. Furthermore,
movements of the paramagnetic center lead to averaging of
the paramagnetic effects, which hampers the translation into
structural information. (iii) The paramagnetic center should
assume a single conformation. Many metal complexes show
flexibility of parts of the coordinating cage, including exchange
of coordinating groups. Different coordination states usually
result in different orientations of the Δχtensor and are thus
prone to producing more than a single set of PCSs or RDCs (in
the slow exchange limit) or line broadening (in the intermediate
exchange regime). (iv) For probes that are linked via two
identical tethers (e.g., to a pair of cysteine residues in the target
protein), C2symmetry is important to avoid that attachment
results in different isomers, each with its own Δχtensor. (v) The
metal affinity needs to be high and the probe needs to be stable
and easily available, either commercially or by straightforward
chemical synthesis.
3.1. Metalloproteins
3.1.1. Paramagnetic Metalloproteins. Among the metal-
loproteins with paramagnetic properties, iron and copper
proteins are the best studied by paramagnetic NMR. Iron
often occurs in either iron−sulfur clusters, such as in
ferredoxin,
113,131
or coordinated to heme rings, like in
cytochromes.
132
In FeS clusters, iron is bound to sulfurs from
cysteine and inorganic sulfur.
62
In heme, the macrocycle acts as a
tetradentate ligand for the iron and provides space for additional
axial ligands. Bertini and co-workers extensively studied iron
proteins by paramagnetic NMR.
112,133−139
The structure of
oxidized Saccharomyces cerevisiae iso-1-cytochrome c, containing
a low-spin Fe(III) ion, was the first paramagnetic metalloprotein
for which the structure was refined with paramagnetic
restraints.
139
The strategy used started with a known structure
based on other restraints, such as NOEs, to fit the Δχtensor of
the paramagnetic center. This allowed using the PCSs as
additional restraints in new rounds of structure calculations and
refinement of the Δχtensor based on the new structure. This
iterative method has proven successful in yielding a more
accurate 3D structure of the target protein.
112,139
3.1.2. Metalloproteins with Paramagnetic Substitu-
tion. In many cases, metals are either not paramagnetic or have
inconvenient paramagnetic properties. In this situation, it may
be possible to substitute the natural metal ion with another one
characterized by different paramagnetic properties. For example,
Cu(II) is usually ligated by histidine, cysteine, aspartic acid, or
tyrosine residues, or a sulfide.
115,140
As Cu(II) mainly generates
PREs, the NMR signals of the coordinating residues are very
broad. By substitution of Cu(II) with Co(II), Donaire et al.
studied coordination in the blue copper protein azurin
141
and
Bertini et al. investigated the metal binding site of
stellacyanin.
142
Also Ln(III) ions have been used for
substitution of metal ions of similar ionic radius and
coordination chemistry.
143
Calcium binding sites have been
particularly successful in this respect and Ln(III) ions have
frequently been successfully substituted for Ca(II) in the EF-
hand motif.
119,144
Most calcium proteins feature a pair of EF-
hand motives, but simultaneous substitution of both Ca(II) ions
with two Ln(III) ions is unfavorable due to electrostatic
repulsion. Therefore, calbindin D9k, which possesses two Ca(II)
binding sites in EF-hand motives, is suitable for selective Ln(III)
substitution into a single one of the Ca(II) binding sites.
145
Using calbindin D9k as a model protein, the whole lanthanoid
group was incorporated in this way, yielding a useful
comparative study of their paramagnetic effects (Table 2).
125
Similarly, calmodulin, which harbors four calcium binding sites
in four EF-hand motives, has successfully been studied by
paramagnetic NMR
146
and the Asn60Asp mutant was shown to
promote the Ln(III) affinity and specificity of a specific Ca(II)
binding site.
145,147,148
3.2. Genetically Encoded Metal Binding Sites
3.2.1. Natural Amino Acids or Peptides. Metal-generated
paramagnetic structure restraints were initially explored in detail
with paramagnetic metalloproteins and, having realized their
exceptional value for 3D structure determinations of proteins,
subsequently extended to diamagnetic metalloproteins by
substitution with paramagnetic metal ions. In nonmetallopro-
teins, paramagnetic metal binding sites can be created by
chemical modification of the target protein. Inspired by the
strategy of substituting Ln(III) ions into EF-hands, lanthanoid-
binding peptides (LBPs) have been proposed for adding a
paramagnetic center to a protein (Table 3). Initially, a LBP with
an EF-hand like motif was fused to the target protein at its N-
terminus.
149
Subsequently, LBPs with improved lanthanoid ion
binding affinity were identified by the Imperiali group and the
number of residues reduced to 17.
150−152
Double-lanthanoid-
binding tags were shown to enable the binding of two Ln(III)
ions simultaneously in a peptide with less than 40 residues.
153,154
Because of the high mobility of terminal fusion tags, however,
the paramagnetic effects in the target protein were greatly
reduced by averaging. This situation was improved substantially
by inserting an LBP into protein loops, as demonstrated for
three different loops of interleukin-1β(IL1β) and confirmation
of the tag structure by X-ray crystallography.
155
Coordinated
with Gd(III) ions, these constructs can also be used for Gd−Gd
distance measurement by EPR spectroscopy.
154
Disadvantages
Table 3. Frequently Used Lanthanoid Binding Peptides
lanthanoid binding peptide sequence refs
DNDGDGKIGADE (“EF-hand”)149
YIDTNNDGWYEGDELLA 151,152,740−742
YIDTNNDGAYEGDELLA 152
YIDTNNDGWIEGDELLA 741,742
YIDTNNDGAYEGDELSG 743
YIDTNNDGWIEGDEL 741
GYIDTNNDGWIEGDELY 154,155
YVDTNNDGAYEGDEL 157,158,626
GDYNKDGWYEELE 150
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K

of this approach are that it limits paramagnetic centers to termini
or loop regions of the protein and any isotope labeling of the
protein also labels the LBPs, which complicates the NMR
spectra especially for the diamagnetic reference. In addition, this
approach requires that the structure of the target protein is
known, while the exact location of the lanthanoid ion is still
difficult to predict in advance.
To overcome these drawbacks, LBPs were developed that can
be attached by linking them to the protein chemically. Generally,
these tags contain free thiol groups for attachment to cysteine
residues.
156
They can be introduced anywhere by introducing a
cysteine residue on the protein surface. Su et al. designed a series
of LBPs with a cysteine residue for attachment to cysteine in the
protein via a disulfide bond.
157
The Δχtensor could be varied by
including either a D-orL-cysteine in the LBP or varying the
position of the cysteine residue in the LBP. Using the N-terminal
domain of the Escherichia coli arginine repressor (ArgN) as
model protein, large paramagnetic effects were obtained for all
Ln(III) loaded LBPs with different tensor orientations for each
of the tags,
157
but the mobility of the LBPs relative to the protein
decreased the anisotropic paramagnetic effects similar to the
LBP fusion method.
154
To reduce the mobility of the LBP tag, it
has been proposed to anchor the tag at two sites.
158,159
The first
double-anchored LBP was designed by Saio et al. and combined
a N-terminal fusion with a chemical linkage to the target protein
via a disulfide bond.
158
Following fusion of the LBP to the N-
terminus of the B1 immunoglobulin binding domain of protein
G (GB1), a cysteine residue in the LBP was linked to a cysteine
residue in GB1 by thiol activation using 5,5′-dithio-bis(2-
nitrobenzoic acid) (DTNB) to form the second linkage. The
double linkage was referred to as L2GB. As expected for a more
rigid tag attachment, larger Δχtensors were observed compared
with the same LBP attached by fusion only (referred to as
L1GB).
158
An alternative straightforward approach is to use the metal
binding propensity of canonical amino acids like histidine,
aspartic acid, and tyrosine to bind metal ions. For example, His6
tags are routinely installed for protein purification and have been
explored also for paramagnetic NMR. Unfortunately, like N-
terminal LBP fusions, this tag proved too mobile to yield good
properties for paramagnetic NMR beyond generating PREs.
160
In contrast, dihistidine motives can generate better defined
metal ion binding sites and have been used for EPR distance
measurements between two Cu(II) ions.
161−163
This approach
was recently extended successfully to paramagnetic NMR
studies, where a Co(II) ion bound to a dihistidine motif
generated sizable PCSs in various proteins.
164
It was shown that
dihistidine motives with good metal binding affinity can be
installed either in α-helices or β-sheets.
164
3.2.2. Noncanonical Amino Acid and Their General
Synthetic Approaches. Well over 100 noncanonical amino
acids (ncAAs) with a wide variety of side chains have been
incorporated into proteins by genetic encoding.
165−167
Among
these, some are capable of binding metal ions. For example,
bipyridylalanine (BpyAla,Figure 4)
168
has successfully been
used to endow proteins with new hydrolytic function or greater
stability enabled by its capacity to bind divalent metal ions.
169,170
Paramagnetic transition metal ions, such as Co(II), which prefer
nitrogen ligands and require fewer coordination sites than
Ln(III) ions, can be coordinated by BpyAla. A West Nile virus
NS2B-NS3 protease (WNVpro) mutant containing a BpyAla
residue in a loop was shown to generate PCSs following
coordination with a Co(II) ion, but additional coordination was
required from proximal side chains, as was demonstrated by
mutation of nearby residues.
171
2-Amino-3-(8-hydroxyquinolin-
3-yl)propanoic acid (HQA,Figure 4)
172
has equally been
explored as the basis for metal ion binding motives.
173−178
Unfortunately, Ln(III) ions coordinated by HQA generally lead
to quantitative protein precipitation,
178
but Mn(II) captured by
HQA has successfully been used to generate PREs in membrane
proteins.
173
A related approach uses site-specific incorporation of one or
two phosphoserine residues, which is a naturally occurring
amino acid produced by post-translational modification, but
which can also be incorporated as a ncAA in response to an
amber stop codon, owing to a recently developed genetic
encoding system.
179−181
A phosphoserine residue in con-
junction with an aspartate or glutamate residue, or two
Figure 4. Chemical structures of some natural (1), noncanonical (2−9,11) amino acids, and nitroxide radicals (10,12).
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L

phosphoserine residues together with a glutamate residue can
generate Ln(III) binding sites that position the metal ion very
precisely on the target protein, generating substantial Δχ
tensors.
182
Unfortunately, the close proximity of charged amino
acid side chains produces significant electrostatic repulsion,
which can lead to unfolding of the protein and poor protein
yields.
As discussed in section 2, not only paramagnetic metal ions
but also nitroxide radicals constitute useful paramagnetic centers
for generating PREs. Noncanonical amino acids containing a
nitroxide radical have been synthesized (Figure 4, compounds
4−7)
183−185
and some of them have been incorporated into
proteins by genetic encoding.
51,183
Alternatively, a nitroxide
radical can be attached to a ncAA after incorporation in the
target protein. In one method, an azido or alkynyl-bearing ncAA
(Figure 4,8a/9a or 8b/c/9b)
186−188
is installed in the target
protein and reacted with an alkynyl or azido-functionalized
nitroxide radical (Figure 4,10a or 10b),
188
using the copper-
catalyzed azide−alkyne cycloaddition (CuAAC) reaction
189
to
form a triazole linker. A similar strategy employs the Suzuki−
Miyaura coupling reaction, where an iodide ncAA (Figure 4,11)
is reacted with a boronic acid spin label (NOBA,Figure 4,
12).
190
For ncAAs containing a nitroxide group from the
beginning, reduction of the radical by the reducing conditions
inside E. coli cells limits the yield of successful incorporation.
Among the nitroxide radical ncAAs mentioned above, only
TOAC was used as a paramagnetic center for NMR studies, to
generate PREs in the complex between a peptide derived from
focal adhesion kinase (FAK) and Src homology 3 (SH3) domain
of Src kinase.
191
Others were only used for EPR stud-
ies.
51,183,188,192−195
Two main strategies are followed in the synthesis of ncAA,
which involve either the side chain modification of a natural AA
or alkylation of a glycine equivalent (Scheme 1).
196−198
Recently, additional strategies for ncAA synthesis have been
published.
199−201
Method A of Scheme 1 is most frequently
applied to synthesize ncAA probes. p-Azido-L-phenylalanine,
which is one of the most popular ncAAs, is synthesized by the
first strategy with L-phenylalanine as the starting compound.
Two approaches give the product in two steps (Scheme
2A).
202−204
Similarly, compound 4is synthesized by coupling
reaction between L-asparagine and a nitroxide radical derivative
(Scheme 2B).
183
BpyAla and HQA are ncAAs that can directly
coordinate metal ions. Their synthetic routes differ from those
mentioned above. Instead of starting from a natural AA, their
synthesis starts from the compounds possessing the metal
binding ability, which are modified with amido and carboxyl
groups to form the final ncAA, as shown in Scheme 2C and
D.
168,172,205
3.3. Synthetic Tags for Proteins
To obtain useful paramagnetic results, other than solvent PREs
or RDCs, it is critical that the paramagnetic center is at a fixed
distance and orientation relative to the nuclear spin and its
molecular frame. Rapid distance and orientation variation lead
to nonlinear averaging of the paramagnetic effects, making
interpretation more complicated or impossible. Thus, the
linkage between the paramagnetic center and the molecule
needs to be as short and rigid as possible, contrary to, for
example, the longer linkers that are often used in fluorescence
spectroscopy. Second, it is critical that a paramagnetic center is
attached to only a single, well-defined site on the molecule. The
presence of more than one center greatly complicates the
interpretation of PCS and PRE data. Similarly, if the tagging site
is undetermined, the information content of PCS and PRE data
becomes uncertain. As a consequence, tagging of proteins is best
achieved on a residue with unique chemical reactivity on the
accessible surface. For this reason, cysteine is by far the most
popular residue type targeted for tagging, as many proteins lack
surface exposed cysteine residues, so that unique sites can be
engineered by site-directed mutagenesis. If exposed cysteines are
present in the native protein, they can be replaced first by alanine
or serine, usually without consequence for the structure or
function of the protein. The general procedure requires
reduction of any disulfide bridges using a reducing agent such
as dithiothreitol, and removal of the reductant, before the
tagging reaction can take place.
3.3.1. Nitroxide Probes. 3.3.1.1. Nitroxide Probes for
Studies of Biomolecular Structure. Nitroxides are extensively
used as spin-labels for structural studies of proteins, protein−
DNA complexes, and protein−RNA complexes by EPR and
NMR spectroscopy. Two main factors need to be considered for
the application of nitroxides in biological systems. The radical
character must be stable with respect to the chemistry used to
attach the tag to the target molecule. Nitroxides are stable under
most nonreducing conditions but can nonetheless be prone to
oxidation to an oxammonium cation or reduction to a
hydroxylamine (see Scheme 3). Several ways have been reported
to stabilize nitroxides.
206
Besides increased chemical stability
associated with five- versus six-membered heterocycles and
bulky substituents adjacent to the nitroxide group, a nitroxide
equipped with cyloalkanyl groups (Figure 5,13) also tends to
possess a longer electronic relaxation time than MTSL (Figure
3).
207
The most efficient way of connecting a nitroxide label to a
biomolecule is to use an activated thiol, such as methanethiosul-
fonate (MST)
208,209
or a pyridylthio group,
210
which form
disulfide bonds with cysteine residues. Disulfide bonds,
however, are easily cleaved under reducing conditions. There-
fore, maleimide
211,212
and iodide
212−214
nitroxide probes have
been developed (Figure 5,14−18) to form thioether bonds with
cysteine residues, which are more stable under reducing
conditions than disulfide bonds.
An important consideration is that any flexibility of the linker
(Figure 6A) leads to averaging of the spin−spin interactions
over a range of distances, reducing the accuracy of any distance
measurements.
215−218
Probe mobility can be restricted by
adding a bulky chemical group on the heterocycle (Figure 5,
19),
219
introducing a second attachment group (Figure 5,20−
22),
220−224
or incorporating a free radical containing unnatural
amino acid into a sterically crowded site of the target protein
(Figure 4,4−7).
183,195
In addition, nitroxides with alternative
reactive functionalities have been reported, such as the azide
Scheme 1. Synthetic Strategies for Modification of Amino
Acids
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M

(Figure 5,23−25)
225,226
and alkyne (Figure 5,26−30)
227−229
containing probes amenable to “click”chemistry for attachment
to, for example, p-azido-L-phenylalanine (AzF, Figure 4,9a).
Others contain an isothiocyanate (Figure 5,31−32)
230,231
or
carbodiimide (Figure 5,33)
231
group for reaction with a
glutamate side chain, the hydroxylamine ether probe (Figure 5,
34)
232
to generate an oxime ether upon reaction with proteins
containing a p-acetylphenylalanine residue, or a hydroxysucci-
nimide ester (Figure 5,35)
207
for reaction with a lysine side
chain. These probes contain bulky groups in the vicinity of the
nitroxide group and are well suited to minimize the movement of
the radical relative to the protein frame.
3.3.1.2. General Synthetic Approaches toward Nitroxide
Probes. Several methods have been established for the
preparation of nitroxides.
206
In general, the nitroxide is formed
by oxidation of a secondary amine with meta-chloroperbenzoic
acid (m-CPBA) or H2O2as oxidant (Scheme 3). The latter
method requires a catalyst. In both methods, the amine group is
first oxidized to form a hydroxylamine intermediate that is
subsequently further oxidized to an oxammonium salt
intermediate to finally yield the nitroxide. Alternatively, a mild
oxidant, such as MnO2, can be applied to oxidize a hydroxyl-
amine to give the desired nitroxide (Scheme 3). The m-CPBA
oxidation is fast because less polar solvents can be used and
compounds with electron-deficient double bonds are tolerated
under these conditions.
233
In contrast, H2O2in the presence of a
catalyst (tungstate salt) requires the use of a polar solvent
system, in which lipophilic substrates are poorly soluble.
Scheme 2. Synthetic Routes of p-Azido-L-phenylalanine (A),
202−204
Compound 4 (B),
183
BpyAla (C),
205
and HQA (D)
174
,
a
a
The functional groups of the ncAAs are colored in blue.
Scheme 3. Two Synthetic Approaches toward Nitroxide Radicals
a
a
(a) Oxidation with m-CPBA or H2O2/cat. Na2WO2. (b) Oxidation with MnO2.
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N

As discussed in section 3.3.1, there are two factors that need to
be considered for improving the stability of nitroxides, one being
the ring size and the other the substituents in the α
positions.
206,234
In the following, we discuss the synthesis of
different types of nitroxides with five- or six-membered rings and
various substituents in the α-positions.
Tetramethylpiperidone (Scheme 4,36), which is commer-
cially available, is the most commonly used starting material for
the synthesis of nitroxide probes, due to its reactive ketone
group. It can readily be modified to provide water-soluble
TEMPO derivatives (see Scheme 4) or embellished with a
reactive functionality (Scheme 4,37a−d).
225,231,235
Sakai et al.
successfully developed a mild method to exchange the
tetramethyl groups of compound 36 by more bulky six−
membered heterocyclic or homocyclic rings (Scheme 4,38a−
b).
236
In this reaction ammonium chloride acts as catalyst and,
by using 15NH4Cl, the authors were able to study the mechanism
of this interesting exchange reaction that appears to involve two
cross-aldol reactions, two fragmentations and two Michael
additions, one of which acts as the final cyclization step.
236
Similarly, the ketone group of compound 38a was transformed
to produce compounds 39a−b(Scheme 4).
225
The sulfur−
carbon bonds of compound 38c can be removed by Raney nickel
to give a tetraethyl substituted derivative (Scheme 4,40).
237
After oxidation of the NH of compound 40 to a nitroxide, the
hydroxyl group can be converted to different functional groups
(compounds 41a−c).
225,238,239
Tetramethylpiperidone is also a precursor for the synthesis of
five-membered ring nitroxides via a Favorskii ring contrac-
tion.
225,240
Tetramethylpiperidone 36 is first brominated to
yield compound 42 or 43. Under basic conditions, compounds
42 and 43 give the pyrrolidine derivative 44 and the pyrroline
45, respectively (Scheme 4). A similar sequence of steps leads to
compounds 46 and 48 (Scheme 4).
225,233,237
Another method based on the Horner−Wadsworth−
Emmons reaction for making α-tetrasubstituted piperidones
from a bisphosphonate has been reported (Scheme 5).
241
In this
procedure, a monoenone (Scheme 5, compound 49) and a
dienone intermediate (Scheme 5,compound50a)were
generated. The monoenone was further reacted to give
nonsymmetric dienone (Scheme 5, compound 50b). Both
dienones (Scheme 5, compounds 50a and 50b) further reacted
with hydroxylamine through a double aza-Michael addition
reaction to yield the corresponding tetra-substituted piper-
idones 51a and 51b, which were oxidized to give the nitroxides
(Scheme 5, compounds 52−54).
Isoindolinyl nitroxides are of special interest due to their
benzannulated scaffold with diminished flexibility as compared
Figure 5. Structures of nitroxide probes. The functional groups for covalent attachment to cysteine via a thioether are highlighted in red and the
functional groups for covalent attachment to a ncAA are highlighted in blue. Probe 13
207
is derived from MTSL; 14
211
and 15
212
are maleimide
functionalized nitroxides; probes 16,
213
17,
213
and 18
391
are iodide functionalized nitroxides; probes 20,
221
21,
221,223
and 22
224
are double-anchored
nitroxides; probes 23,
225
24,
225
and 25
225
are azide functionalized nitroxides; probes 26,
227
27,
227
28,
227
29,
228
and 30
229
are alkyne functionalized
nitroxides; probes 31
230
and 32
231
are iso(thio)cyanide functionalized nitroxides; probe 33
231
is a carbodiimide functionalized nitroxide; probe 34
232
is a hydroxylamine ether functionalized nitroxide; probe 35
207
is a nitroxide functionalized with a hydroxysuccinimide ester.
Figure 6. (A) Reaction of MTSL with a cysteine residue of a protein
generates a flexible linkage with five rotatable bonds (red arrows). (B)
Schematic representation of two different enantiomeric EDTA
complexes produced by different coordination of the metal ion. M
denotes the metal ion. O denotes the carboxyl groups. Curved lines
represent the ethylene groups and dashed lines trace the octahedral
coordination.
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O

to aliphatic nitroxide derivatives.
225
N-benzylphthalimide
(Scheme 6, compound 55) is the most popular starting material
for the synthesis of isoindolinyl nitroxides reported in the early
1980s, via the use of excess Grignard reagent to give 57.
242
A
more efficient procedure than a quadruple Grignard addition
was found later, which proceeded via isolation of the
monoaddition product (compound 56) and subsequent
conversion into compound 57 (Scheme 6).
243
The further
transformation of the benzylamine to a nitroxide was achieved
by debenzylation and oxidation (Scheme 6,58).
244−246
The
latter compound can readily be transformed into a probe, such as
compound 59a/b (Scheme 6), which can be attached to a thiol
Scheme 4. Preparation of Various Nitroxides from Tetramethylpiperidone
a
a
(A) i. NaBH4,H
2O2/cat. Na2WO2(to give 37a);
225
ii. reductive amination with NaBH3CN (to give 37b);
231
iii. active amine with
methanesulfonyl to substitute with NaN3(to give 37c);
225
iv. carboxylation with tosylmethylisocyanide (to give 37d).
235
(B) Cyclohexanone,
NH4Cl, H2O2/cat. Na2WO2(to give 38a−c).
236
(C) Raney−nickel (to give 40).
237
(D) i. Bromination (to give 42,43); ii. Favorskii rearrangement
(to give 44,45).
225,240
Blue colored arrows indicate the synthetic route to five-membered ring nitroxides.
Scheme 5. Synthetic Route to α-Tetrasubstituted Piperidone from a Bisphosphonate Precursor
a
a
(a) i. LDA (lithium diisopropylamide) at 0 °C. ii. BuLi (butyllithium) at −35 °C. iii. excess 3-pentanone. (b) i. LDA, THF, ii. acetone. (c) i.
NH4OH at 105 °C. ii. m-CPBA. (d) Na2WO4,H
2O2. (e) i. TMSCHN2((diazomethyl)trimethylsilane), BF3·OEt2; ii. Na2WO4,H
2O2..
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P

group of a biological macromolecule.
244
Several other routes
toward functionalized isoindolinyl nitroxides have been
reported. One route involves bromination of the aromatic ring
and removal of the benzyl protection group of compound 55,
followed by substitution of the bromide by a cyano or carboxyl
group (Scheme 6,59c/d).
247
Another route involves the
construction of the functionalized aromatic ring via a Diels−
Alder reaction on a diene nitroxide
221
or following a higher
Scheme 6. Synthetic Routes Towards Isoindolinyl Nitroxide
a
a
(a) RMgBr. (b) Excess RMgBr. (c) i. Pd−C/H2; ii. m-CPBA.
Figure 7. Structures of aminopoly(carboxylic acid) based probes. Functional groups for covalent attachment to a cysteine residue in the target protein
via thioether formation are highlighted in red. Alkyne groups for attachment to an azide group in a ncAA via “click”reaction are highlighted in blue.
Probes 60−65
249−253
are EDTA based. Probe 61 has two chiral centers. Probe 66
255
is double-anchored. Probes 73−77
263−266
are PyMTA based. The
asterisk identifies one of the chiral carbon atoms.
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Q

yielding stepwise procedure using a disubstituted pyrroline
nitroxide as starting material, leading to compound 59d.
247
3.3.2. Aminopoly(Carboxylic Acid)-Based Probes.
Aminopoly(carboxylic acid)s have the ability to bind metals
strongly and they can easily be modified with functional groups
to enable linkage to biomolecules. In this section, the most
commonly applied aminopoly(carboxylic acid) based probes
and their synthetic strategies are discussed.
3.3.2.1. EDTA-Based Probes. EDTA (ethylenediaminetetra-
acetic acid) is a widely used chelating agent for a broad range of
metal ions, due to its high metal ion binding affinity with four
carboxylic acids and two amine groups. Its complexes with
paramagnetic ions can be linked to proteins via a disulfide bond
by functionalizing them with MTS or pyridylthio groups. The
first EDTA based paramagnetic probe for proteins, SPy-EDTA
(Figure 7,60), was reported by Ebright et al. in 1992.
248
This
probe soon became commercially available and its various metal
ion complexes were used for protein paramagnetic NMR
studies.
249−251
These studies revealed two sets of PCSs
generated by probe 60 loaded with Co(II) or Dy(III), as the
metal complex produces two enantiomeric forms, which leads to
diastereomers after linkage with the protein (Figure 6B).
249,251
Subsequent introduction of chiral centers and a rigid attachment
group to Ln(III)-EDTA resulted in single sets of PCSs, but the
Δχtensor was relatively small (Figure 7,61).
251,252
Diaster-
eomer formation is not a critical problem for DEER distance
measurements and Su et al. (2015) designed EDTA based EPR
probes with short linkers as Mn(II) tags (Figure 7,62−65),
which yielded a narrow distribution of distances.
253
3.3.2.2. DTPA-, DTTA-, and TAHA-Based Probes. Dieth-
ylenetriaminepentaacetic acid (DTPA) has a structure similar to
EDTA, but features a higher metal binding affinity.
254
DTPA
functionalized with two MST groups, CLaNP-1 (caged
lanthanoid NMR probe 1, Figure 7,66), was designed as an
NMR probe for proteins, using Yb(III) and Y(III) as
paramagnetic and diamagnetic metal ions, respectively.
255
CLaNP-1 loaded with Yb(III) generated large PCSs, but
multiple sets of PCSs were observed for each nuclear spin
observed in the protein, because the coordination cage assumes
multiple conformations.
255
DTPA coordinates lanthanoids in an
octadentate fashion and forms a pair of enantiomers.
256
In 2020,
Su and co-workers showed that a combination of DPTA and L-
cysteine (Py-L-Cys-DTPA (Figure 7,67)
257
yields an open
chain lanthanoid chelator that shows no exchange between
conformations on the low millisecond time. Upon attachment to
various sites on ubiquitin, large PCS and RDC were obtained,
which were dependent on the site of attachment, likely due to a
different degree of mobility for this single-arm tag. The probe
Cys-Ph-TAHA (Figure 7,68)
258
was based on C3-symmetric
TAHA (triaminohexaacetate). The paramagnetic properties of
lanthanoid loaded Cys-Ph-TAHA were determined by using
two ubiquitin mutants (T12C and S57C). PCSs and RDCs were
observed. Two minor species were observed for Cys-Ph-TAHA
labeled ubiquitin T12C, but not for ubiquitin S57C.
258
Two DTTA (diethylene-triamine-tetraacetate) based protein
probes (DTTA-C3-yne and DTTA-C4-yne,Figure 7,69 and
70)
259
were reported, which possess an alkynyl group for “click”
ligation to a genetically encoded ncAA containing an azide
group, with formation of a triazole linker (Figure 4,9a). Tb(III)
or Tm(III) loaded probes conjugated to ubiquitin mutants
generated single sets of PCSs with a good correlation between
experimental and back-calculated values. The triazole ring in the
linker limits the number of rotatable bonds as it can form a
conjugated double-bond system with the phenyl ring of the
noncanonical residue side chain. DTTA-C3-yne, which has a
shorter linker, showed a larger Δχtensor than did DTTA-C4-
yne.DTTA-C3-yne was used in the structure determination of a
transient protein complex.
259
Chen et al. reported phenyl-
sulfonated pyridine modified DTTA probes (Figure 7,71−72),
which can react with cysteine with formation of a thioether bond
shown to be stable in a cell lysate.
260
4PS-6M-PyDTTA (Figure
7,72) has an extra methyl group in position six of the pyridine
ring. This small difference proved to alter the speed of probe
ligation, lanthanoid ion binding affinity, and Δχtensor. 4PS-
6M-PyDTTA showed a higher reactivity toward ubiquitin S57C
and the Staphylococcus aureus sortase A (SrtA) mutant D82C.
260
Scheme 7. Synthetic Route to Probes 71 (4PS-PyDTTA) and 72 (4PS-6M-PyDTTA).
260
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R

Following attachment, 6M-PyDTTA exhibited a lower metal
binding affinity but larger Δχtensor than PyDTTA.
Interestingly, the two probes loaded with Tm(III) gave PCSs
of opposite sign, indicating different orientations of the Δχ
tensor.
260
3.3.2.3. PyMTA-Based Probes. In contrast to the linear
structure of EDTA, 2,2′,2″,2‴-((pyridine-2,6-diylbis-
(methylene))bis(azanetriyl))tetraacetic acid (PyMTA) has a
pyridine core with diazanediyldiacetic acid pendants in positions
2 and 6. Its Ln(III) complexes have been used for luminescence
experiments and as contrast agents in MRI.
261,262
4MMPyMTA
(Figure 7,73) was designed for chemical stability and
successfully used as a paramagnetic lanthanoid probe to study
the structures of minor protein species.
263
4V-PyMTA (Figure
7,74)
264
was reported as a paramagnetic Ln(III) probe with a
vinyl group in position 4, which forms a thioether bond with
cysteine. After successful attachment of 4V-PyMTA to
ubiquitin, small PCSs were observed.
264
Following substitution
of position 3 of the pyridine ring with bromide to obtain probe
75,the reactivity was improved, but not the paramagnetic
effects.
25
Further work introduced phenylsulfonated in pyridine
position 4 as the protein conjugation group (Figure 7,76).
265
Compared with probes 73 and 74, probe 76 yielded larger PCSs
due to the short and rigid thioether attachment, but with a low
reaction rate, even under fairly harsh conditions.
265
Sub-
sequently, probe 77 (Figure 7)
266
was constructed, which
features both good reactivity and selectivity for solvent exposed
cysteine residues.
3.3.2.4. General Synthetic Strategies of Aminopoly-
(Carboxylic Acid)-Based Probes. There are two main strategies
for the synthesis of aminopoly(carboxylic acid) based probes.
The first involves either the direct modification of the carboxylic
acid with attachment groups by a coupling reaction, such as
applied for probes 60 (derived from EDTA) and 66 (derived
from DTPA), or functionalization of the central amine with an
attachment group as in, for example, probes 69 and 70 (derived
from protected DTTA). The second strategy starts from the
core structure of the probe, introduces the attachment group,
and subsequently modifies the intermediate with a polyamine to
introduce the carboxylic acid pendants. For example, the
synthesis of probes 71 and 72 starts from a mono- or
dimethylpyridine derivative, to which the attachment group
(benzenesulfonyl) is added after several modification steps.
Thus, after N-oxidation, nitration, rearrangement to their
trifluoroacetate esters, and in situ mild hydrolysis of α-picoline
or 2,6-lutidine, a nucleophilic aromatic substitution provided the
sulfones. By alkylation with a halogenated pyridine derivative
(Scheme 7, structure colored in red), the polyamine
intermediate (Scheme 7, structure colored in magenta) is
generated after deprotection and finally the poly(carboxylic
acid)s are introduced to give the probe (Scheme 7, structure
colored in blue).
260
3.3.3. Cyclen-Based Tags. Cyclen(1,4,7,10-tetraazacyclo-
dodecane) is a cyclic ring molecule of tetraethyleneamine, which
can be modified to obtain various metal binding complexes,
which are particularly suited for lanthanoid ions. DOTA
(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) is
one of the most frequently used cyclen derivatives. It was first
synthesized in 1976.
267
DOTA complexes are thermally stable
and bind Ln(III) ions tightly.
268
It is important to note that
Ln(III)-DOTA complexes undergo conformational exchange
involving cyclen ring flips combined with reorientation of the
tetracarboxylic acid pendants (Figure 8). The pendant arms can
coordinate clockwise (Λ) or anticlockwise (Δ) and the cyclen
ring has two conformations (λλλλ and δδδδ). Consequently,
Ln(III)-DOTA forms two pairs of enantiomeric species (square
antiprism, SAP, and twisted square antiprism, TSAP) in
solution, which tend to produce different magnetic effects in
the case of anisotropic paramagnetism and averaging between
them due to conformational exchange.
269−271
Therefore, the
designs of the cyclen probes described below aim to lock the
cyclen ring in a single conformation to obtain uniform
Figure 8. (A) Schematic representation of four different stereoisomers populated by DOTA lanthanoid complexes and their conformational exchange.
The cyclen ring is drawn as a square with solid lines; nitrogen, oxygen, and carbon atoms are shown as blue, red, and black spheres, respectively; the
metal ion is represented by a brown sphere. (B) Newman projections showing the effect of the ring flip on the positions of the hydrogen atoms of two
neighboring carbon atoms. Adapted from Q. Miao (2019) Design, synthesis and application of paramagnetic NMR probes for protein structure
studies. PhD Thesis, Leiden University.
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S

Figure 9. Chemical structures of cyclen-based paramagnetic probes. The functional groups for attachment to cysteine via thioether formation are
highlighted in red. The functional groups for attachment to an azide group in ncAA via “click”reaction are highlighted in blue. The cyclen rings with
substituents are shown in magenta. 78,
272
79,
127,273
80,
276
81,
277
82,
278
83,
279
and 84
280
are double-armed probes. Asterisks identify chiral carbon
atoms.
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T

paramagnetic effects, by incorporating substituents on the
cyclen ring and/or the pendant arms with the appropriate
chirality.
3.3.3.1. Double-Anchored Attachment. The first reported
cyclen based protein paramagnetic probe was CLaNP-3 (Figure
9,78)
272
in complex with paramagnetic Ln(III) ions. By
functionalizing two opposing tetracarboxylic acid arms of
DOTA with MTS groups, CLaNP-3 can be anchored to a
protein via two disulfide-bridges for maximum immobilization
of the metal ion relative to the protein. For pseudoazurin (Paz)
with mutations E51C and E54C tagged with Yb(III)-CLaNP-3,
PCSs were detected for nuclear spins up to 35 Å from the
paramagnetic center. Some residues, however, displayed peak
doubling, suggesting that the complex populated more than a
single conformation with different Δχtensors.
272
Subsequently, CLaNP-5 (Figure 9,79)
127,273
was produced
and became one of the most used lanthanoid probes. In CLaNP-
5, the two acetate pendant arms of CLaNP-3 were replaced by
pyridyl-N-oxides, which disfavors the population of the TSAP
conformation of its lanthanoid complexes. For Yb(III)-CLaNP-
5linked to Paz E51C/E54C, a single set of large PCSs was
observed. In contrast, CLaNP-5 attached via a single arm
yielded only small PCSs due to averaging caused by the flexibility
of the attachment.
273
Further studies demonstrated that
magnetic properties of CLaNP-5 are insensitive to the chemical
environment of the attachment site, allowing the prediction of
the Δχtensor parameters and Ln position for a known protein
structure with reasonable accuracy.
127
CLaNP-5, thus, is highly
suitable for structural studies of proteins and protein complexes.
It turned out, however, that the detection of little populated
protein states by relaxation dispersion (RD) experiments is
compromised by the presence of minor conformations of the
complex that can lead to probe-induced RD effects.
274,275
Other
disadvantages of CLaNP-5 are its charge (+3), making it less
favorable for some proteins and protein complexes, and the
disulfide bonds formed with the protein are liable to reduction,
which can lead to loss of the probe over a period of several weeks.
To decrease the charge, CLaNP-7 (Figure 9,80)
276
contains
two phenolic groups, lowering the net charge to +1 by
deprotonation of the phenol hydroxyls. CLaNP-7 is yellow in
color, which is convenient during the tag labeling reaction and
protein purification. Similar to CLaNP-5, it induced a single set
of PCSs in Paz E51C/E54C, with a Δχtensor that was only
slightly smaller than that of CLaNP-5.
276
Unexpectedly, two sets
of PCSs were observed specifically for Yb(III)-CLaNP-7 linked
cytochrome cand this peak doubling was pH-dependent. By
mutation of His39, which is adjacent to the probe attachment
site, to alanine, it was demonstrated that the peak doubling was
caused by an interaction with this residue, suggesting that the
hydration water molecule, which coordinates the Ln(III) ion in
its ninth coordination site, forms a hydrogen bond with the
imidazole ring of His39 and in this way breaks the C2symmetry
of the probe. The effect was pH dependent due to protonation
and deprotonation of the imidazole ring. Since disulfide bonds
can easily be reduced, CLaNP-9 (Figure 9,81)
277
was designed
as a variant of CLaNP-5, featuring two bromoacetanilide groups
to react with cysteine residues by formation of chemically stable
thioether bonds. Using T4 lysozyme (T4Lys) N55C/V57C and
Paz E51C/E54C as model proteins, Yb(III) loaded CLaNP-9
yielded single sets of PCSs under reducing conditions, indicating
the high stability of this probe. Because of the rapid hydrolysis of
Figure 10. Comparison of Co(II)-TraNP-1-SS and Yb(III)-CLaNP-5 attached to T4Lys K147C/T151C. (A) Metal positions and tensor
orientations. (B) PCS iso-surfaces (±0.4 ppm for Yb(III)-CLaNP-5,±0.2 ppm for Co(II)-TraNP-1-SS). (C) Models of the protein−probe
structures. The protein is shown in ribbon representation; the probes and cysteine residues are shown as sticks and the metal ions as spheres.
Reproduced without changes from ref 280. Copyright 2019 the authors, under CCA license (https://creativecommons.org/licenses/by/4.0/).
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U

the bromoacetanilide groups in aqueous solutions, however,
quantitative tagging proved difficult to achieve.
The tags T1 and T2 (Figure 9,82)
278
similarly are double-
anchored cyclen based probes using pyridylthio groups as
attachment groups. T1 and T2 are enantiomers, as their two free
coordination arms are chiral isopropanol moieties. Large Δχ
tensors were found for these lanthanoid complexes linked to
Staphylococcus aureus 6-hydroxymethyl-7,8-dihydropterin pyro-
phosphokinase (HPPK) K76C/C80, and exceptionally narrow
distance distributions were obtained by DEER measurements
using the tags loaded with Gd(III).
278
The paramagnetic effects
induced by other paramagnetic Ln(III) ions, however, are
sensitive to the pH, with large PCSs and RDCs detected at high
pH. Another pair of enantiomeric probes, T7 and T8 (Figure 9,
83),
279
were designed with two shorter chiral attachment arms.
Although large Δχtensors were determined for a wide range of
pH values for both tags, the χtensor orientation changed
between high pH (8) and low pH (6.5). It was noted that T7
produced incomplete ligation yields for one of the test proteins
(HPPK K76C/C80).
279
The tags described above are all lanthanoid based cyclen
probes. Recently, several double-arm 3d block ion probes
(transition metal ion NMR probes, TraNPs) were reported.
280
Among them, TraNP-1 (Figure 9,84)
280
can tightly bind
Co(II) and Mn(II) ions. Its Co(II) complex generates a single
set of PCSs and yields a medium sized tensor, which is suitable
for application in small proteins or localized studies in larger
proteins or protein complexes. The authors compared the tensor
orientation between Co(II)-TraNP-1 and Yb(III)-CLaNP-5
ligated to T4Lys K147C/T151C by Δχtensor fits using
experimentally measured PCSs. The tags delivered significantly
different tensor orientations as evidenced by the PCS iso-
surfaces shown in Figure 10.
280
Recently, the double-anchored probe CLaNP-13 (Figure 9,
85) was designed for DEER studies in living cells.
281
By virtue of
two maleimide groups, CLaNP-13 forms thioether bonds with
cysteine residues, which in a cellular environment are more
stable than disulfide bonds. As Michael additions of thiols to
maleimides generate enantiomers, NMR measurements showed
peak doubling for the Yb(III) complex attached to T4Lys
double-cysteine mutants. DEER measurements, however,
yielded a narrow distance distribution for CLaNP-13 attached
to the quadruple mutant T4Lys N55C/V57C/K147C/T151C,
and the probe was also shown to be suitable for in-cell DEER
measurements.
3.3.3.2. Single Site Attachment. DOTA-M8 (Figure 9,
86)
282
is a single-arm lanthanoid probe, which produces Δχ
tensors of remarkable size. Its structure is based on
M4DOTMA.
283
Because of the eight chiral methyl substitutions
on the cyclen ring and acetate pendant arms, M4DOTMA
loaded with Yb(III) exhibits a single conformation, which is
rigidified by steric crowding.
283
A set of large PCSs was reported
for Dy(III)-DOTA-M8 labeled ubiquitin S57C. Dy(III) is
tightly bound to DOTA-M8, withstanding harsh chemical and
physical conditions.
282
About 15−20% of a minor species
appeared in the NMR spectra, however, and this ratio increased
to 50% by heating the tagged protein to 323 K. Subsequent
studies revealed slow conformational exchange between two
coordination geometries of Ln(III)-DOTA-M8, which produce
different Δχtensors, with the exchange rate depending on the
radius of the lanthanoid ion.
271,284
As DOTA-M8 is ligated to the protein via a disulfide bond, the
ligation is not stable under reducing conditions. This prompted
efforts to link DOTA-M8 to proteins by different attachment
chemistries. By replacing the pyridylthio linker arm in DOTA-
M8 by a propyliodoacetamide (−C3H6NHCOCH2I) group, a
chemically more stable thioether bond is formed in the reaction
with cysteine. This new tag was referred to as M8-CAM-I
(Figure 9,87) and its usefulness for in-cell NMR measurements
was demonstrated, but the longer linker resulted in a smaller Δχ
tensor than for DOTA-M8.
285
By using 4-(phenylsulfonyl)-
pyridine as the attachment group, another DOTA-M8
derivative, referred to as M7Py-DOTA (Figure 9,88), forms a
more rigid and shorter linkage to the protein, which is also
suitable for in-cell NMR.
286
As the reactivity of the 4-
(phenylsulfonyl)pyridine group is relatively low, the CF3-
substituted phenyl analogues (Figure 9,89 and 90) were
designed for enhanced reactivity, but this approach did not
increase the rate of protein tagging because the rate-limiting step
appears to be the formation of a Meisenheimer-type complex
rather than the dissociation of the pyridine-sulfone moiety.
287
Consequently, the probes M7FPy-DOTA (Figure 9,91)
287
and
M7PyThiazole-DOTA (Figure 9,92)
287
were produced, both
of which proved to react with cysteine with high selectivity and
efficiency. The authors found that M7FPy-DOTA can react also
with tris(2-carboxyethyl)phosphine (TCEP) to release benze-
nesulfinic acid, which can catalyze protein dimerization by
intermolecular disulfide bridge formation between cysteine
residues. M7PyThiazole-DOTA performs better, as it is more
reactive toward cysteine than TCEP and generates an even
larger Δχtensor than DOTA-M8.
287
Recent work introduced a
3-nitro-substituted 2-(methylsulfonyl)pyridine moiety as at-
tachment group to design a new DOTA-M8 based probe named
M7-Nitro (Figure 9,93).
288
This probe possesses high protein
ligation rates (more than 95% ligation in 30 minutes at room
temperature) and a very large Δχtensor (Δχax = 94.3 ×10−32
m3) was measured with Dy(III)-M7-Nitro labeled ubiquitin
S57C. The authors attributed the improvements in ligation to
the methylsulfone leaving group in the ortho position and the
presence of the nitro substituent in the meta position.
To further improve the rigidity of the cyclen-based probes,
DOTA derivatives with more crowded substituents, P4M4-
DOTA (Figure 9,94)
126
and P4T-DOTA (Figure 9,95)
289
were synthesized. Only a single conformation, Λ(δδδδ), was
detected in ROESY spectra of the Lu(III)-P4M4-DOTA
complex.
126
Linking lanthanoid ion loaded P4M4-DOTA to
ubiquitin S75C and human carbonic anhydrase II (hCA II)
S166C delivered single sets of PCSs, but some of the Qfactors of
the Δχtensor fits were relatively large owing to the length and
flexibility of the attachment arm. As expected, P4T-DOTA
performs better than P4M4-DOTA because of a shorter and
more rigid linker. P4T-DOTA also forms a stable thioether
bond with cysteine.
C1 (Figure 9,96) is another early example of a DOTA based
probe for protein studies by paramagnetic NMR, which is
relatively easy to synthesize.
290
In this tag, the metal ion is
coordinated by amides instead of carboxyl groups. Loading of
C1 with Ln(III) ions requires harsh conditions and the ions are
tightly bound thereafter. Three chiral bulky pendant arms ensure
that the C1 tag populates a single conformation as indicated by
1H NMR spectra of Yb(III) loaded C1 and single sets of PCSs
were observed with all proteins tested. C2 (Figure 9,96) is the
enantiomer of C1 and generates a slightly different tensor
orientation.
291
On the basis of the structure of probe C1, the
protein EPR probe C9 (Figure 9,97)
292
was designed with a
chiral tagging arm. Variants of C1 with alkyne attachment
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V

groups, C3 and C4 (Figure 9,98,99),
293
were developed for
ligation to the ncAA AzF (Figure 4,9a)by“click”chemistry.
293
The PCSs obtained with probe C4 are smaller than those of
probe C3 due to the longer linker of the former. The Cu(I)
catalyzed ligation reaction was shown to be selective also in the
presence of cysteine residues in the target protein and thus
suitable for proteins containing cysteine residues of structural
and functional importance. Subsequent experience showed,
however, that many proteins are prone to precipitation in the
presence of Cu(I) catalysts.
182
For this reason, the recently
published C12 probe (Figure 9,100)
294
reacts with cysteine by
formation of a thioether bond to its aromatic ring linkage group.
Complete tagging yields of cysteine were obtained at room
temperature overnight. In contrast, the tag reacts with
selenocysteine in minutes, providing a route to selective tagging
of proteins in the presence of native cysteine residues.
294
Ln(III) complexes of C1 and its variants are relatively large,
and although experimental evidence is scarce,
295
their hydro-
phobic aromatic rings may result in unexpected specific
interactions with the protein surface. Therefore, a number of
alternative cyclen probes were produced with hydrophilic
pendants, where the single conformation of the cyclen ring is
ensured by the chirality of the pendant arms. The probe C5
(Figure 9,101)
129
contains three chiral (S)-2-hydroxypropyl
groups as pendant arms. Variants of C5 with different
attachment linkers were also explored, including C6 (Figure 9,
102),
129
which contains a rigid picolinic acid linker, C7 (Figure
9,103),
129
which contains a chiral alcohol in the attachment arm
instead of an amide, and C8 (Figure 9,103),
129
which is the
enantiomer of C7. Like C1 and C2, these tags can generate large
Δχtensors. A comparison of three proteins and different
attachment sites showed, however, that the Δχtensor produced
by C7 is highly sensitive to the protein environment, which is
unusual for lanthanoid based cyclen probes.
129
Probe C10
(Figure 9,104)
296
was designed for DNA studies (discussed in
section 3.4). Ligated to ubiquitin A28C, it produced Δχtensors
of medium size, as expected for the relatively long and flexible
attachment arm. Recently, DO3MA-Py (Figure 9,
105),
297
DO3MA-6MePy (Figure 9,106),
297
DO3MA-3BrPy
(Figure 9,107),
298
and BrPSPy-6M-DO3M(S)A (Figure 9,
108)
299
were reported, which contain methyl substituted
carboxylic acid pendants and phenylsulfonated pyridine with
different substituents on the pyridine ring as functional group for
attachment. These lanthanoid ion complexes carry zero net
charge and form thioether bonds with cysteine residues. Large
PCSs were observed with these probes. While the ligation rates
of DO3MA-Py and DO3MA-6MePy are slow even at high pH
and temperature, the bromide substituent in DO3MA-3BrPy
decreased the reaction time to 6 h at ambient temperature.
Sizeable Δχtensors were obtained with the Yb(III) complex of
DO3MA-3BrPy attached to ubiquitin D39C and ubiquitin
E64C, and DO3MA-3BrPy loaded with Gd(III) was also
Scheme 8. Three Strategies for Cyclen Ring Modulation
a
a
The magenta colored structures show the final cyclized ring with different substituents. The compounds in magenta are applied for paramagnetic
probe design.
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W

successfully used for in-cell distance measurements by EPR,
although the distance distribution obtained was relatively
broad.
298
Its companion BrPSPy-6M-DO3M(S)A has an
extra methyl group on the pyridine and the authors reported
that this design improved the rigidity of the tag.
300
By replacing
bromine in DO3MA-3BrPy for a fluorine atom, Su and co-
workers synthesized the probes 4PS-5F-Py-DO3MA (Figure 9,
109) and 4PS-5F-Py-DO3A (Figure 9,110) to analyze the
conformational exchanges of DOTA-Ln-like probes using 1D
and 2D EXSY NMR spectroscopy.
301
They demonstrated that
even the presence of chiral centers in three of the pendants of a
DO3A-type cyclen tag does not prevent the population of a
minor conformational species, which persists following ligation
of the lanthanoid complexes to ubiquitin G47C.
301
Notably, a
second conformational species has also been reported for
DOTA-M8, although this probe contains four additional
stereocenters in the cyclen ring.
287
The relative conformational
stabilities of SAP and TSAP conformations of DOTA-type
cyclen-Lu(III) complexes with different chiralities of methylated
cyclen and carboxylate pendants has recently been rationalized
computationally.
302
Another recent approach used a photocatalyzed reaction to
conjugate a vinyl probe to proteins.
303
Using 2,2-dimethoxy-2-
phenylacetophenone (DPAP) as a photoactivated radical
initiator, the ligation rate of the vinylpicolinic acid modified
probe 111 (Figure 9) with GB1 T53C was greatly accelerated,
but the Δχtensors obtained were small.
303
3.3.3.3. General Synthetic Approaches of Cyclen Based
Probes. DOTA and its analogues have been used as metal ion
ligands for decades and the thermodynamic properties,
synthesis, and applications of their metal complexes have been
investigated in detail.
268,304−308
Commensurate with the wide
range of applications for cyclen and DOTA compounds in many
different fields, numerous synthetic routes have been reported. It
should be noted that DOTA based probes with chiral
substituents to ensure conformational rigidity, require the
execution of a multistep synthetic scheme. In addition, the
protective group strategy has to be chosen carefully to allow the
introduction of one or two attachment arms. Among the
different synthetic methods reported for large-scale cyclen
preparation,
307,309−314
three main strategies stand out for
modification of the cyclen ring. The first is based on one of
the synthetic procedures of cyclen (Scheme 8A). This approach
was explored by Jacques et al., who used substituted ethylenedi-
amine derivatives and chloroacetyl chloride derivatives as
starting materials to form a linear diamide intermediate (1c in
Scheme 8A), which was further reacted with an ethylenediamine
derivative to yield the cyclic product 1d. Finally, the amide
groups of the ring were reduced to give the core macrocyclic
structure 113 (Scheme 8A).
315,316
On the basis of this method,
symmetric and asymmetric functionalized cyclen derivatives
were produced, such as cyclohexyl, cyclohexene and tetraline
substituted cyclens 113a−c,
304
and the diol substituted
compounds 113d−e.
315,316
Later on, the same group developed
a new method to synthesize cyclen derivatives with C4
symmetry, where, instead of using a stepwise cyclization, a
more straightforward synthetic route was followed by
oligomerization of N-substituted aziridine (2a in Scheme 8B)
in the presence of p-toluenesulfonic acid (PTSA, Scheme
8B).
283,317
This method directly gives the N-protected cyclen
derivatives (2b in Scheme 8B), albeit in low yield, and, after
deprotection, the desired macrocycle 114. The cyclen based
probes DOTA-M8,M7Py-DOTA,M7FPy-DOTA,P4T-
DOTA as well as M7PyThiazole-DOTA (Figure 9) were
synthesized using this approach and several other groups
subsequently used this approach for cyclen based ligand
synthesis.
313,314
Anelli et al. employed both approaches for the
synthesis of more hydrophilic macrocyclic compounds.
318
Figure 11. Chemical structures of small molecule probes. Functional groups designed for attachment via thioether formation are highlighted in red.
116,
322
117,
323
118,
324
121,
326
122,
326
and 124
265
are DPA-based probes. 119 and 120 are TDA based probes. 123
176
is based on 8-hydroxyquinoline.
125
327
is an NTA based probe. 126
328
and 127
329
are IDA based probes.
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X

Recently, Häussinger and co-workers reported a new method for
synthesizing modified cyclens (Scheme 8C),
126,319
which starts
from chiral aminoaldehydes (3c in Scheme 8C) derived from
their respective amino acid precursors. Via reductive amination,
two suitably protected linear alkylated amino acid derivatives
(3e and 3f) are produced, which are readily coupled to each
other and cyclized by standard solution peptide coupling under
high dilution. Compared to the other two strategies, this newly
reported route provides a high yield in the cyclization step and
the overall yield of macrocycles 115 has been reported around
22%.
319
3.3.4. Small Molecule Tags. Small molecules like
dipicolinic acid (DPA), iminodiacetic acid (IDA), nitrilotri-
acetic acid (NTA), and [2,2’:6′,2″]terpyridine-6,6″-dicarboxylic
acid (TDA) have also been explored for the design of
paramagnetic NMR probes. Their attraction derives from their
small size, which minimizes the potential impact on the protein
structure, and their relative ease of synthesis. In complexes with
Ln(III) ions, these small probes leave several coordination sites
of the Ln(III) ion free for coordination by solvent molecules or
additional protein side chains. With these tags, the metal
complexes are formed by titration with metal ions after probe
attachment, which opens the possibility of inaccurate metal to
protein ratios and binding of metals to other, undesired sites.
3.3.4.1. DPA, TDA, and HQA Tags. DPA coordinates metal
ions in nonchiral geometry, thus reducing the potential for
different enantiomeric forms of metal coordination and peak
doubling in NMR spectra. Despite presenting only a tridentate
ligand, the affinity of DPA for lanthanoid ions is in the low
nanomolar range.
320,321
4MMDPA (Figure 11,116)
322
was the
first DPA tag developed. It was attached to a cysteine residue
following activation with DTNB, which results in a disulfide
linkage, and shown to generate PCSs following attachment to
Cys68 of the ArgN protein, in which the coordination of the
paramagnetic lanthanoid ion appears to be supported by
coordination to a glutamate side chain.
322
Despite the limited
coordination of the lanthanoid ion, the dissociation rate of the
metal ion was slow at 25 °C (0.1 s−1), but the Δχtensor was
smaller than that found with cyclen based tags. Loaded with
Gd(III) ions, the tag was also demonstrated to be suitable for
Gd−Gd distance measurements by EPR spectroscopy in
different proteins.
122
To shorten the linker length, the probes
3MDPA (Figure 11,117)
323
and 4MDPA (Figure 11,118)
324
were produced, which feature the sulfur atom right on the
pyridine ring. 3MDPA, which has a thiol group in position 3, was
ligated to ArgN like 4MMDPA via a disulfide bond following
activation of its cysteine residue with DTNB. In complex with
paramagnetic Ln(III) ions, Δχtensor orientations were
obtained different from those with 4MMDPA and metal
coordination still appeared to be assisted by Glu21, but the
magnitudes of the Δχtensors were small. Compared to the
3MDPA tag, the disulfide dimer of 4MDPA is a more
convenient tag for ligation, as it reacts spontaneously and
quantitatively with cysteines by disulfide exchange. While it
displayed relatively small Δχtensors with ArgN, larger Δχ
tensors were obtained with the intracellular domain of the p75
neurotrophin receptor (p75 ICD). The tensor orientations
differed substantially from those obtained with the correspond-
ing 4MMDPA complexes for ArgN but not for p75 ICD,
suggesting important contributions by additional coordinating
amino acid side chains. The DPA tags also display good affinity
for Co(II), as demonstrated first for ArgN with 3MDPA tag.
323
Using DPA as starting materials, TDA based probes, 4MTDA
(probe 119,Figure 11)
325
and 4MMTDA (probe 120,Figure
11)
325
were synthesized. Large PCS were detected after their Ln
ion loaded complexes were ligated to ubiquitin T22C or A28C.
They also exhibit fluorescent properties. For both mutants,
ubiquitin side chains are involved in metal coordination. The
authors also showed that 4MTDA exchanges lanthanide ions
more slowly than DPA, as 2D 1H−1H EXSY spectra showed no
observable chemical exchange. The ligation yields were reported
to be relatively low.
Probe 121 (Figure 11)
326
contains a vinyl group to link the
probe to the protein via Michael addition to form a thioether
linker. The vinyl group avoids the problem of maleimide
functionalized tags (Figure 11,122), which create a new chiral
center upon reaction with a thiol group.
326
A vinyl group was
also used to ligate the 8-hydroxyquinoline based probe 2V-8HQ
(Figure 11,123)
176
to a cysteine. While the tag is not suitable for
binding lanthanoid ions, it binds divalent transition metal ions
such as Mn(II), Co(II), Ni(II), Cu(II), and Zn(II) with
micromolar affinities, and a large Δχtensor was observed for the
Co(II) complex of 2V-8HQ bound to ubiquitin A28C/E24H,
where the binding of the metal ion was assisted by the histidine
residue.
176
A DPA derivative with a phenylsulfonated pyridine
functional group (Figure 11,124)
265
was shown to generate a
stable, rigid and short tether with a thioether bond to a cysteine,
but Δχtensor parameters were not reported.
3.3.4.2. NTA and IDA Tags. Similar to DPA and 8HQ tags,
NTA-based probes immobilize metal ions on target proteins
depending on the assistance from additional coordinating
groups provided by other amino acid residues. The first NTA-
based probe suitable for binding lanthanoid ions, NTA-SH
(Figure 11,125),
327
was obtained by modifying cysteine with
two acetate groups. Single sets of PCSs were reported upon
complexation with paramagnetic lanthanoid ions following
attachment of the probe to either one or two cysteine residues of
the target protein. For a single NTA-SH tag, much larger Δχ
tensors were observed with ArgN than ubiquitin A28C,
suggesting that the protein environment plays a critical role in
immobilizing the Ln(III) ion. This difficult-to-predict variability
can be reduced by simultaneously attaching NTA-SH tags at
positions of iand i+ 4 of an α-helix to create a metal binding site
with coordination by both NTA moieties. This approach can
also deliver very large Δχtensors.
327
As the metal ion affinity is
not as high as for DOTA ligands, the metal ion can be exchanged
by washing with EDTA and subsequently introducing another
metal ion.
An even smaller tag is based on IDA, which only contains two
carboxylates. The IDA-SH tag (Figure 11,126)
328
and its
activated form (Figure 11,127)
329
were reported to yield a
single set of PCSs after chelating lanthanoid ions. This tag can
yield large Δχtensors, if the Ln(III) ion can be additionally
coordinated by a protein side chain as observed for ubiquitin
A28C, where Asp32 is located in the same α-helix as residue
28.
328
By ligating two IDA-SH tags to cysteine residues in
positions iand i+ 4 of an α-helix, a Co(II) ion can be
coordinated in an octahedral complex and produces sizable
PCSs (Figure 11,127).
329
In ubiquitin E24C/A28C, however,
this tag arrangement performed best with substoichiometric
quantities of Co(II) and Zn(II) ions, whereas lanthanoid ions
produced multiple species. Subsequent study of this tag and
metal complexation mode in the same protein and in T4Lys
K147C/T151C revealed additional species also with Co(II)
ions and difficulties to achieve complete saturation of the metal
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binding site.
280
A systematic exploration of the best binding
partner for an IDA-SH or NTA-SH tag attached to a cysteine
residue in position iof an αhelix indicated that the IDA-SH tag
forms the best Ln(III) binding sites together with an aspartate
residue in position i+ 4, while the sterically more demanding
NTA-SH tag produces the best sites in combination with a
glutamate residue in position i−4.
330
As secondary structure
predictions identify amphipathic αhelices with good reliability,
this approach is of interest for proteins of unknown 3D structure.
3.3.4.3. Hybrid Double-Arm Attachment. Nitsche et al.
recently introduced a new tagging strategy, where a small metal
binding tag is attached to two cysteine residues, but additional
coordination of the lanthanoid ion by a negatively charged side
chain of the protein is still required to immobilize the metal
ion.
331
The concept was demonstrated with the Zika virus
NS2B-NS3 protease mutated to contain cysteine residues in
positions 101 and 131. The side-chains of the cysteine residues
are in close proximity and avidly bind the pnictogens arsenic,
antimony, and bismuth in oxidation state III. The third
coordination site of the pnictogen is available for binding a
mercaptomethylaryl tag, such as 4MMDPA to capture a Ln(III)
ion, which was shown to generates sizable Δχtensors in
conjunction with additional coordination by a glutamate
residue. The fundamental advantage of the concept is its
selectivity for two cysteines in close proximity. As single thiol
groups bind pnictogens with negligible affinity, it allowed
tagging of the protein in the presence of the native, solvent
exposed single cysteine residues.
3.3.5. Cosolute Paramagnetic Probes. Cosolute para-
magnetic NMR probes do not chemically modify the targeted
biological macromolecule. A structurally diverse class of
paramagnetic molecules has been designed and investigated
for their noncovalent interaction with the solvent exposed
surface residues of oligopeptides and proteins. Cosolute probes
can be as simple as salts of paramagnetic transition metals. The
approach is based on the idea that large compounds and charged
Figure 12. Chemical structures of nitroxide based cosolute probes.
Figure 13. Chemical structures of metal ion based cosolute probes.
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Z

ions do not readily access the interior of folded proteins, so that
the NMR signals of surface residues experience stronger PREs
than buried residues.
332
Mn(II) ions are well suited for this type
of NMR experiments.
333−335
Owing to its diradical character, molecular oxygen is
paramagnetic and one of the simplest molecular probes used
in NMR. As oxygen dissolves more easily in the hydrophobic
interior of membranes than in aqueous solution, this solubility
gradient has been used to probe the structure and positioning of
a membrane protein,
336
and a number of related NMR
experiments have been reported that exploit the unique
properties of oxygen.
337−341
In early work, Williams and co-
workers obtained cosolute paramagnetic effects by directly
adding paramagnetic metal ions, such as Gd(III), Eu(III), and
Mn(II), to protein solutions.
110,120,342
Using [Cr(CN)6]3−,
[Fe(CN)6]3−, and [Co(CN)6]3−complexes in 1D 1H NMR
spectroscopy experiments, Williams and co-workers studied the
competition with negatively charged substrate molecules for
binding to phosphoglycerate kinase.
111
Organic nitroxide radicals present a more varied class of
paramagnetic cosolute probes. Early results showed that di-tert-
butylnitroxide generates contact shifts with several small organic
molecules in carbon tetrachloride.
343
On the basis of these
results, Kopple and Schamper
344
prepared 3-oxyl-2,2,4,4-
tetramethyloxazolidine (Figure 12) that is soluble in chloroform
and methanol, and these solvents and this probe were used to
detect line broadening in the 1H NMR spectrum of amides of the
solvent exposed amino acid residues in the C2symmetric cyclic
decapeptide gramicidin S, while the amides of the amino acid
residues involved in intramolecular H-bonding were not
affected. The same research group reported that a water-soluble
variant (HyTEMPO or TEMPOL,Figure 12) can serve as a tool
to identify hydrophobic surface residues of proteins, using the
attenuation of antiphase COSY cross-peaks as a sensitive
method to detect small line-broadening effects caused by the
presence of the paramagnetic agent.
345
Building on these results,
Fesik and co-workers showed that quantitative measurement of
R1(1H) relaxation rate enhancements generated by TEMPOL
allowed accurate identification of the solvent exposed regions of
cyclosporin A bound to cyclophilin.
346
TEMPOL also proved
useful to probe the accessibility of amino acid residues in protein
unfolding experiments
347
and studies of conformational
change.
348
The attempt to probe electrostatic potentials using
the charged derivatives 4-carboxy-TEMPO and 4-amino-
TEMPO (Figure 12) showed the expected trends but also
demonstrated limitations attributed to local variations in
translational diffusion constants.
347−349
Stronger solvent PREs
can be generated by Gd(III) ions and the uncharged and highly
water-soluble Gd(III)DTPA-BMA probe (Figure 13) can
perform better than TEMPOL, because it is less hydrophobic,
stable with regard to redox-active compounds, and the
paramagnetic center less accessible.
350
Solvent PREs generated
by Gd(III)DTPA-BMA were shown to deliver powerful
restraints for 3D structure determinations of proteins and
peptides,
35,351,352
which prompted the design of Gd(III)-
TTHA-TMA
353,354
as a more spherical Gd(III) probe without
Figure 14. Most relevant chemical methods to connect NMR or EPR probes to one or more cysteine residues in proteins. The aim of the figure is to
provide an overview of the currently used chemistry for the modification of cysteine residues, suitable for the attachment of a chemically sensitive
paramagnetic center. The use of maleimide as reactive group results in the generation of a chiral center, and therefore, this type of linkage is not
recommended in NMR studies.
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AA

any hydration water bound to the Gd(III) ion. The use of
alternative uncharged Gd(III) probes such as Gd2(L7)-
(H2O)2,
355
Gd(DO3A), and Gd(HP-DO3A),
356
as well as
complexes with increasing charge, such as [Gd(EDTA)]1−,
357
[Gd(DTPA)]2−,
358
and [Gd(DOTP)]5−,
359
has also been
explored (Figure 13). The strong PRE effects generated by
Gd(III) complexes have also proven useful to shorten the T1
relaxation time of H2O
360
and protein protons
361
without
greatly increasing the line widths of protein NMR signals,
enabling sensitivity enhancements by faster scan repetition rates.
It is important to note, however, that some proteins may harbor
specific binding sites for some of these complexes, which can be
made apparent by PCSs generated by complexes with
paramagnetic lanthanoid ions other than Gd(III). For example,
the cosolute probe [Tb(DOTP)]5−was found to generate
specific PCSs in the DNA binding protein fd gene 5
359
and
[Ln(DPA)3]3−(with Ln = Tm, Yb, or Tb, Figure 13) was found
to generate significant PCSs in ArgN and several other
proteins.
362,363
The Δχtensor generated in this way can be
tuned by using DPA derivatives carrying different substituents
(−Br, −COOH, or −CH2OH) on the aromatic ring, which form
lanthanoid complexes like the parent [Ln(DPA)3]3−and bind to
the same site on the protein.
363
As rapid reorientation of the
complexes averages PCSs to zero,
350
the observation of PCSs
can be taken as positive proof of specific binding. Natural
binding sites for [Ln(DPA)3]3−are infrequent in proteins and it
has been shown that binding sites specific for this complex can
be engineered by positioning the side chains of two positively
charged residues (lysine or arginine) in close proximity, which,
at the same time, need to be sufficiently far from negatively
charged carboxylates to prevent their engagement in salt
bridges.
364
In contrast to covalently bound paramagnetic
probes, specific binding sites for complexes such as [Gd-
(DPA)3]3−offer the opportunity to measure PREs for nuclear
spins very close to the binding site, as the PRE magnitude can be
tuned by the amount of relaxation agent added.
365
PREs
generated by the cyclen complexes Gd(DO3A),Fe(DO3A),
and Ni(DO2A) (Figure 13) have also been explored. The
Gd(III) ion in Gd(DO3A) has two accessible coordination sites
available and specifically targets carboxylates on protein
surfaces.
366
Fe(DO3A) has been used to speed up data
acquisition by shortening the T1relaxation time of an
intrinsically disordered protein.
367
Ni(DO2A) has been
reported to be particularly suitable for shortening the T1
relaxation times of H2O and protein signals without broadening
them significantly.
368
As the ligand fills all coordination sites of
the Ni(II) ion, it is unlikely to bind to specific sites on proteins.
At the same time, it is a water-soluble probe without carrying a
net charge.
3.3.6. Development of Cysteine Based Probe Attach-
ment Chemistry. Historically, the development of chemical
approaches for the selective modification of proteins has its
origin in studies of amino acid chemistry.
369
Further important
insights on site-specific chemical modification came from
studies of post-translationally modified proteins.
370
New
chemistry for site-specific ligation reactions were opened by
the development of methods to incorporate noncanonical amino
acids either by genetic encoding or the use of chemically
misacylated tRNAs.
371−373
The thiol group of cysteine has unique nucleophilic
characteristics, as compared to the side chains of any of the
other canonical amino acid residues. Because of the higher
acidity of RCH2SH as compared to RCH2OH, cysteine residues
in proteins can be deprotonated at slightly basic pH, which
increases their reactivity
374−383
toward soft electrophiles. With
little chemical activation of the paramagnetic probes, the
reaction conditions used for the attachment of the probes can
be mild. Figure 14 provides an outline of the most relevant
cysteine modifications with respect to the attachment of probes
amenable for NMR and EPR.
384−386
A standard, selective and mild, method for attaching a probe
to a cysteine residue is by disulfide formation. One of the most
frequently used reactive functionalities to accomplish this is the
MTS or S-mesyl group. This group is stable under acidic
conditions, which is advantageous, if acid labile protective
groups need to be removed at a late stage of chemical probe
synthesis.
280
At physiological pH the thiol of cysteine forms a
disulfide linkage with the RS-moiety of the S-mesyl compound
with the release of methane and SO2in a practically irreversible
reaction. Several other reactive functionalities are known to
generate S−S bonds,
387
in particular the 2-pyridyldisulfanyl
group (as in the probes 60,
248
82,
278
83,
279
86,
282
94,
126
96,
290
97,
292
101,
129
103,
129
112,
388
and 127
329
). Disulfide bonds are
also made by activation of a cysteine residue of a protein using a
mild oxidizing agent such as Ellman’s reagent (DTNB) and
addition of the probe as a thiol.
322,323,327,328,389
Similarly, a
chelating moiety was introduced by reaction of a cysteine
residue with ArS-SAr (Ar = PyMTA
266
or Ar = DPA
324
). Several
procedures are available to produce a thioether, which is
chemically more stable than a disulfide linkage. α-Iodo or α-
bromo carbonyl reagents (XCH2C(O)R) have traditionally
been used to generate thioethers with cysteine residues, as the
reaction is fast and selective at the physiological pH.
369,390
This
reactive functionality has successfully been installed in a number
of probes, such as in probes 16,
213
17,
391
81,
277
87,
285
and
104.
296
Furnishing DOTA type probes with two α-bromo-
carbonyl functionalities, however, has proven challenging
because of the high reactivity of this functionality.
277
Another standard reactive functionality is the maleimide
group, which has a high reactivity and selectivity for cysteine
residues around neutral pH.
392,393
Maleimide probes react with
cysteines via Michael addition, forming a new stereo-
center
211,212,281
and thus giving rise to different stereoisomers
which complicate PCS assignments. The use of a maleimide
functionality to attach a probe for NMR experiments is therefore
not recommended. Monobromomaleimide probes
223,394
do not
have this problem because the Michael addition is followed by
an elimination reaction resulting in a thio-enol ether linkage
(Figure 14), and Michael reactions with vinylyridine probes
(probes 64,
253
65,
253
74,
264
75,
25
121,
326
and 123
176
) do not
generate new chiral centers either. Tosylpyridine probes (62,
253
63,
253
71,
260
72,
260
76,
265
77,
266
88,
286
89,
287
90,
287
91,
287
93,
288
105,
297
106,
297
107,
298
108,
300
109,
301
110,
301
and
124
265
) form thioethers. The electrophilicity of these probes
is enhanced by the presence of a halide substituent on the pyridyl
ring. Similar to the tosyl group, a nitro group on the pyridine ring
can act as a leaving group.
294
3.4. Paramagnetic Probes for DNA and RNA
Studying oligonucleotides with paramagnetic probes can
provide valuable insights into the structure and dynamics of
DNA and RNA as well as their interactions with proteins.
40−47
Labeling methods of oligonucleotides can be classified into four
main categories. First, noncovalent spin labels can be selectively
introduced at an abasic site without covalent attachment of the
probe to the nucleotide. Second, spin labels can be selectively
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attached to the nucleotide backbone via reaction with modified
ribose or phosphate groups in the oligonucleotide. Third, spin
labels can be selectively attached to a single base via reaction
with a modified purine or pyrimidine base. Fourth, a fully
synthetic and already labeled nucleotide can be introduced into
oligonucleotides by standard solid-phase oligonucleotide syn-
thesis. Occasionally, a synthetic method may allow the
attachment of probes to the nucleotide prior to, during, or
post oligonucleotide synthesis. All four strategies are reviewed in
the following sections with a particular emphasis on the
attachment chemistries.
395
3.4.1. Noncovalent Spin Labels. Paramagnetic cosolutes,
which have been used intensively for the study of proteins
(section 3.3.5), have also been used to study oligonucleotides by
paramagnetic NMR spectroscopy. For example, the nitroxide
radical TEMPOL (Figure 12) and the Gd(III) complex of
TTHA-TMA (Figure 13) have been used to study the surface
accessibility of RNA and refine its 3D structure.
396,397
To probe biomolecules more precisely, it is necessary to
introduce the spin label at a defined position as rigidly as
possible. In the case of proteins, this is usually achieved by site-
selective mutation and subsequent covalent bond formation
between probe and amino acid side chains (section 3.3).
Selective and rigid noncovalent labeling of proteins is not
straightforward and achievable only in special cases, requiring
the availability of a suitably labeled protein ligand.
398
In the case
of oligonucleotides, noncovalent spin labeling is a much more
generally applicable technique that appeals by its simplicity.
Synthetic spin labels can mimic a purine or pyrimidine base and
specifically bind to an abasic site in double-stranded DNA or
RNA oligomers, thus circumventing the need to synthesize the
entire spin-labeled nucleotides. Currently available noncovalent
spin labels are all nitroxide radicals, which allow applications in
NMR (PRE) and EPR. Noncovalent oligonucleotide tags
containing a paramagnetic metal ion may be suitable for PCS
measurements but are yet to be explored.
The first site-directed noncovalent spin label, which binds to
an abasic site in DNA, was reported by Lhomme and co-workers
(Figure 15,128).
399,400
Compound 128 combines the base
adenine with the DNA intercalator acridine, which are
connected via a flexible linker. Nakatani and co-workers
developed spin label NCD-TEMPO (Figure 15,129) and a
nitronyl nitroxide analogue NCD-NN (Figure 15,130), which
can specifically bind to G−G mismatches in DNA duplexes,
Figure 15. Site-directed noncovalent spin labels for DNA and RNA.
Scheme 9. Synthesis of the Noncovalent DNA Spin Label çfrom 5-Bromouracil
a
a
5-Bromouracil is regioselective benzylated, subsequently converted into its O4-sulfonylated derivative (TPS = 2,4,6-triisopropylbenzenesulfonyl)
and coupled to an isoindol aminophenol derivative, followed by ring-closure. The benzyl group is removed and N-oxidation gives the nitroxide.
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allowing for the construction of tandem arrays and three-
dimensional assemblies of electron spins.
401−404
Spin label NA-
TEMPO (Figure 15,131) was designed in this work to clarify
the effect of orthogonal sequence selectivity on the electron-spin
arrangement.
402
Sigurdsson and co-workers introduced the noncovalent DNA
spin label ç, which allows labeling without intercalation or
specific sequence requirements.
405
Its synthesis from 5-
bromouracil is shown in Scheme 9. Mimicking a cytosine, ç
binds best to a DNA duplex if paired with a guanosine nucleotide
(Kd∼5μMat−10 °C).
405
Binding of ç(Figure 15) does not
only depend on the pairing base, but also on the flanking
sequence, with a 5′-d(DçT) sequence affording the highest
affinity.
406
The abasic site should also be located at least four
base pairs from the end of the DNA duplex.
406
A DNA duplex
labeled with two çlabels enabled DEER distance measurements
up to 70 Å. Binding of a double-stranded lac operator DNA
sequence to the lac repressor protein has also been probed by
DEER. Bending of the double helix was manifested in a distance
decrease between both çspin labels by about 5 Å, which,
however, is close to the resolution limit of this method.
407
The
introduction of substituents at the N3 nitrogen of çaffected the
affinity, with large substituents compromising the affinity
most.
406,408
Derivatives containing an amino or guanidino
group displayed slightly increased affinity and improved
solubility.
408
The anucleoside C3(Figure 15) was explored as
an alternative abasic site over the tetrahydrofuran analogue F
(Figure 15) with limited impact on the affinity of çto the
DNA.
406
The nitroxide radical 132 (Figure 15) was developed as an
alternative noncovalent spin label that binds to abasic sites in
RNA and DNA duplexes.
409
Best pairing was observed with
cytosine which does not interact well with ç. Although spin
labeling of RNA and DNA was confirmed by EPR spectroscopy,
132 has not been used in any further applications. Another
campaign screened ten pyrimidine based spin labels for their
suitability to occupy abasic sites in DNA and RNA duplexes.
410
Triazole linked (e.g., 133 (Figure 15)) and pyrrolocytosine-
based (Figure 15,134) labels showed very promising results.
Spin label 133 displayed increased solubility and affinity (DNA)
compared to its N1-methylated analogue (not shown). It was
speculated that the increased affinity may result from a salt
bridge formed between the ethylamino group and the phosphate
backbone. The DNA label çhas insufficient affinity to RNA
(∼30%).
410
In contrast, 133 shows much higher affinity (100%
under identical conditions) to an abasic RNA site; it also binds
to an unmodified RNA duplex, which, however, compromises
specificity.
410
The isoindoline-nitroxide derivative G
́overcomes previous
limitations and binds with high affinity and specificity to abasic
sites in RNA duplexes. G
́pairs with a cytosine and is suitable for
DEER distance measurements when combined with an RNA
duplex containing two abasic sites.
411
G
́can be prepared from
commercially available materials in a single straightforward
synthetic step (Scheme 10), facilitating its broad use.
411
G
́can
also bind to abasic sites in DNA-RNA hybrids, either in the RNA
or DNA strand, and, similar to ç, must be positioned at least four
residues from the end of the duplex to achieve optimal
binding.
412
The adenine derived spin label 135 complements
the guanidine derived G
́(Figure 15); 135 was identified from a
series of purine derived nitroxides and can be introduced at
abasic sites in DNA and RNA duplexes where it pairs with
thymine or uracil, respectively.
412
G
́has been successfully
applied to measure distances in both DNA and RNA duplexes by
DEER.
413
Combined with molecular dynamics simulations,
these experiments indicate strong Watson−Crick base pairing,
no disturbance of the overall helical structure (minor local
disturbances occur) and rigid binding of G
́. Recently, G
́was also
used to label DNA and RNA triplexes containing a single abasic
site.
414
The N-oxide phenazine 136 is the latest noncovalent
spin label that can bind to an abasic site in DNA but is unsuitable
for RNA.
415
If the spin label can directly bind to RNA or DNA, no
chemical modifications of oligonucleotides (not even the
introduction of an abasic site) are required. Despite the principal
appeal of this strategy, however, achieving the necessary
specificity of the interactions between label and oligonucleotide
can be challenging, as observed for intercalating spin labels
based on chlorpromazine,
416
ethidium bromide, or other
polyaromatic carcinogens,
417
Ru(II)-phenanthroline com-
plexes,
418
or acridine.
419
Bifunctional cross-linking agents are
also of limited specificity.
420,421
One solution is presented by the
use of an aptamer. For example, the malachite green RNA
aptamer (a 38-nucleotide sequence) is known to bind malachite
green or closely related dyes such as tetramethylrosamine
(TMR). If TMR is synthetically combined with a spin label as in
137 (Figure 15), it can be used to selectively label the RNA
aptamer.
422
Combination with a spin label that is covalently
attached to the RNA backbone (145, see section below) allowed
DEER measurements. Similarly, a ternary complex consisting of
a DNA oligomer, the molecule chromomycin-A3and para-
magnetic Co(II), where Co(II) was bound to two oxygens of the
chromophore, has been used to measure PCSs and improve the
structure of the complex.
423,424
3.4.2. Probe Attachment to the Sugar−Phosphate
Backbone. Some paramagnetic spin labels can be attached to
the phosphate backbone of DNA and RNA. It has been noted
that spin labels attached to phosphodiesters interfere less with
duplex formation than other labels, as they are located at the
edges of the polynucleotide helices.
43
The greatest disadvantage
of this approach is that labeling of phosphodiesters inevitably
results in the formation of diastereomers, which need to be
separated by chromatography and identified, if unambiguous
structural information is to be obtained from paramagnetic
NMR experiments or EPR studies.
296,425,426
Recently, a
synthetic strategy for the chiral synthesis of phosphorothioates
Scheme 10. Synthesis of the Noncovalent RNA Spin label G
́from 2-Bromohypoxanthine via a Nucleophilic Aromatic
Substitution
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has been described, which may help to overcome these
challenges.
427
In addition, it is important to note that, when
labeling RNA, the 2′hydroxyl group adjacent to the labeled
phosphodiester needs to be protected as a 2′-OMe group or
replaced by a hydrogen atom to prevent strand cleavage through
2′,3′-transesterification.
428
The introduction of a hydrogen phosphonate group during
oligonucleotide synthesis enables the selective attachment of 4-
amino-TEMPO via H-phosphonate chemistry yielding a
phosphoramidate (Figure 16A).
429,430
Using protected oligo-
nucleotides, 4-hydroxy-TEMPO can be attached to the 3′or 5′
end of DNA via a phosphoester bond (not shown),
431
which has
been used to study protein DNA interactions by NMR.
432
Similarly, the increased reactivity of terminal phosphate groups
can be exploited by selective activation of the 3′and 5′ends of
unprotected DNA and subsequent reaction with 4-amino-
TEMPO to form phosphoramidates.
433
A fully protected
TEMPO-phosphoramidite building block (Figure 16B, 138)
compatible with oligomer synthesis has been used to form an
ester between 4-hydroxy-TEMPO and the phosphate at the 5′
end of RNA.
434
A frequently used functional group for site-directed labeling of
the phosphate backbone is presented by phosphorothioate.
Selective incorporation of spin labels at the 5′end of RNA is
possible directly by transcription from DNA in a two-step
process where, first, a DNA template is transcribed in the
presence of NTPs (nucleotidetriphosphates) and a phosphor-
othioate nucleotide and, second, a spin label is attached to the 5′
phosphorothioate group. The approach has been demonstrated
with guanosine monophosphorothioate (139), T7 RNA
polymerase, and spin label 140, which was attached via disulfide
formation (Figure 16C).
435
Alternatively, a phosphorothioate moiety can be enzymati-
cally installed in the 5′position of RNA or DNA using T4
polynucleotide kinase, prior to alkylation with the spin label 16
(Figure 16).
436
The iodoacetamide based spin label 17 (Figure
16) can be ligated to internal phosphorothioate nucleotides in
DNA (Figure 16E).
437
The rigid spin label 16 has been used
more frequently.
438
It has been attached to RNA and DNA to
study, for example, the GAAA tetraloop/receptor interaction,
439
the human telomeric G-quadruplex,
440
riboswitch dynamics,
441
CRISPR-Cas9,
442
DNA structure and dynamics,
443
distances by
DEER,
444
and protein DNA interactions.
445
The 4-bromo-
substituted analogue R5a has been used to study ribozymes,
CRISPR-Cas9, and the structure and dynamics of
DNA.
426,446−449
The double-alkylating analogue R5c links two
adjacent phosphorothioates to give a conformationally highly
constrained cyclic spin label for RNA and DNA (Figure
16D).
220
Graham and co-workers developed the first lanthanide
tag (Figure 9,C10) that can be attached to phosphorothioate,
enabling the observation of PCSs in NMR spectra of DNA.
296
Alternatively, paramagnetic spin labels can be attached to the
sugar (ribose) part of the backbone. The 2′position in
oligonucleotides is the only position readily available for
modification. Nucleotides bearing a 2′amino group can be
site-specifically installed in oligonucleotides using standard
oligonucleotide synthesis (2′amino-modified phosphoramidites
of pyrimidine-based nucleotides are commercially avail-
able).
450,451
The most commonly used spin label is 4-
isocyanato-TEMPO (Figure 17,142), which is readily
accessible from 4-amino-TEMPO and diphosgene.
450,452
The
Figure 16. Strategies for the attachment of probes to the phosphodiester backbone or terminal phosphate groups of oligonucleotides.
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isocyanate group forms a stable and rigid urea moiety between
the 2′position of the nucleotide and the TEMPO spin label,
which has been used in numerous applications involving EPR
and NMR to study RNA and its interactions.
453−461
An
alternative succinimide-based spin label (Figure 17,143) was
found to affect the thermostability of RNA helices.
462
It can also
be introduced at a 5′amine or piperazine group (Figure 17).
463
The isoindoline-based spin labels 144 (Figure 17) and 145
(Figure 17) bear an isothiocyanate group to form a thiourea
linkage with the 2′amino group.
230
The tetraethyl derivative
145 (Figure 17) displayed significant stability toward reducing
conditions.
Attaching a probe to the 2′position via “click”chemistry has
also been studied extensively (Figure 18). Attachment of probe
24 (Figure 18)toa2′propargyl ether was the first example
described; however, the construct produced broad distance
distributions in DEER experiments due to a rather long and
flexible linker.
43,464
A much shorter linker can be established, if
the alkynyl group is directly connected to the 2’carbon as in 2′-
alkynylnucleotides (Figure 18).
225,465
Azide based spin labels
23−25 (Figure 18) and 146−148 (Figure 18) can be attached
selectively via “click”chemistry and have been used to measure
distances in DNA by DEER.
225
A crystal structure of double-
stranded DNA with label 24 has been solved (Figure 19).
465
Combined with EPR experiments and MD simulations, the
results indicate only minimal perturbance of the DNA structure
because of the presence of the spin label. Royzen and co-workers
developed alkyne substituted Cu(II) (Figure 18,149) and
Co(II) (Figure 18,150) binding ligands that can be attached to
a linker-azide functional group in the 2′position (Figure
18).
466,467
Both tags were successfully used to study binding of
the HIV-1 nucleocapsid protein 7 to an RNA pentanucleotide by
PRE measurements using NMR spectroscopy.
466,467
In the case
of the cobalt tag 150, observation of PCSs was not reported,
which potentially relates to the flexibility of the linker between
probe and RNA.
467
The “click”reaction between a 2′azido GTP
and an alkyne-TEMPO derivative produces a spin labeled
nucleotide that can be used for site-specific post-transcriptional
Figure 17. Probe attachment to (A) a 2′amino group of internal nucleotides (blue arrows indicate the labeling reactions) and (B) alternative 5′
modifications.
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labeling of RNA (not shown).
468
The GTP analogue is a
substrate for a Tb3+-deoxyribozyme that installs it specifically on
an internal adenine nucleotide via a 2′,5′-phosphodiester
linkage.
468
Finally, spin labels can be selectively attached to the 5′or 3′
hydroxy ends of nucleosides. Selective 5′labeling can be
achieved via carbamate or amide formation with TEMPO,
430
143 (Figure 17B),
463
or the large trityl radical 151 (TAM)
(Figure 20).
469
Attachment to both 5′nucleotides of a DNA
duplex allows distance measurements by DEER. Although trityl
radicals offer advantages over nitroxide radicals, such as narrow
spectral width and stability under reducing conditions,
43
their
size and hydrophobicity limit applications with biomolecules.
Recently, alternative linkers were developed that allow the
attachment of TAM anywhere in the DNA sequence using an
achiral non-nucleoside phosphoramidite (Figure 20).
470
Selective modification of the 3′end of RNAs with TEMPO
has also been demonstrated.
471−473
3.4.3. Probe Attachment to Modified Bases. The
majority of paramagnetic probes used to study RNA and DNA
are attached to purine or pyrimidine bases. Labels can be fully
introduced by synthetic chemistry to furnish labeled nucleotides
that can subsequently be used directly in solid-phase
oligonucleotide synthesis (covered in the next section).
Alternatively, labels can be introduced postsynthetically (by
chemical synthesis or transcription) by attachment to a modified
base either prior or post removal of protection groups (in the
case of chemical synthesis). The postsynthetic approach can
help minimize possible decomposition of the nitroxide radical
during synthesis, but it may result in incomplete labeling or side
reactions with other functional groups.
43
A functional group frequently employed for postsynthetic
modification is presented by sulfur substituents in purine and
pyrimidine bases (Figure 21). 4-Thiouracil has been used most
frequently. 4-Thiouridine can be found in natural tRNAs of E.
coli and has been selectively labeled with spin label 17/141
(Figure 16) without the need for any synthetic modifications.
474
Subsequently, 4-thiouridine was installed in synthetic RNA
and labeled with 17/141 to study a double-stranded RNA-
binding domain in the Staufen protein by NMR spectroscopy.
475
A similar protocol
476
was also used to determine the solution
structure of large RNAs and protein RNA complexes,
477
for
example, to study E. coli protein sequestration by the noncoding
RNA RsmZ by NMR and EPR.
478
A very similar system using
deoxy-4-thiouridine and 17/141 in synthetic DNAs (installed at
a5′overhang) allowed the study of DNA and the modulator
recognition factor 2 (Mrf2) by NMR measurements of PREs.
479
As an alternative to the formation of thioethers, thiouridine can
form disulfide bonds with spin labels 152−154 (Figure 21),
which has been used to study RNA structures.
480
2-Thiocytidine
has been incorporated into tRNA using tRNA nucleotidyl
Figure 18. Probe attachment via “click”chemistry to the 2′position.
Figure 19. X-ray crystal structure (PDB code 6QJS) of a 12 base pair
DNA duplex containing the spin label 24 (highlighted in pink) attached
in the 2′position via “click”chemistry. The phosphate backbone is
shown as a ribbon. The distance between both nitroxide radicals
determined by DEER is 30 Å.
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transferase and reacted with spin label 155.
481
The rare
nucleotide 2-thio-5-(N-methylaminomethyl)-uridine from the
anticodon region of E. coli tRNAGlu was acylated with spin label
156.
482,483
The thiopurine nucleotide 6-thioguanosine can be
enzymatically incorporated into RNA via a DNA-splint
mediated ligation strategy and subsequently reacted with spin
label 17 to study long RNAs by NMR spectroscopy (Figure
21).
484,485
Convertible nucleotides present yet another strategy to
introduce a functional group during oligonucleotide synthesis
that can selectively react with spin labels postsynthetically. This
strategy uses aromatic ethers or fluorine as leaving groups and
amino groups in spin labels, such as 4-amino-TEMPO,as
nucleophiles (Figure 22).
486−489
Both pyrimidine and purine
analogues have been described, and the approach has been
employed in several studies involving RNA and DNA.
490−495
Alkynyl bases, such as the commonly used 5-ethynyl-2′-
deoxyuridine (EdU), have been developed to allow for the
selective reaction with azido based spin labels (e.g., 24,25,148)
via “click”chemistry during postsynthetic modification of DNA
and RNA (Figure 23A).
496−503
The tags have been demon-
strated to be particularly useful for distance measurements by
Figure 20. Attachment of the triarylmethyl (TAM)-based spin label 151 to the 5′end of DNA. The immobilized oligonucleotide is treated with CDI
and piperazine and cleaved from the support using standard conditions. The resulting oligonucleotide is treated with cetyltrimethylammonium
bromide (CTAB), taken up in DMSO and treated with 151 as the chloride in the presence of base. The two remaining acid chloride functions were
hydrolyzed. Two alternative TAM attachments strategies are shown.
Figure 21. Postsynthetic probe attachment to thiopyrimidines and thiopurines.
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DEER.
496,497,499,500,502,503
The strategy also enables the labeling
of very long RNAs (>400 nucleotides) and distance measure-
ments within them.
500
NHS esters 143 and 159 (Figure 23B)
have been used to acetylate nucleotides that contain aliphatic
amines (Figure 23B).
504−507
Selective labeling of guanine
involves a multistep process including the use of a comple-
mentary DNA reagent. Despite the length of the resulting
linkers, the strategy successfully enabled DEER distance
measurements in RNA duplexes. An analogue of cisplatin
(160,Figure 23C) has also been used to deliver spin labels to
DNA by ligand exchange with guanine (Figure 23C),
508
enabling paramagnetic NMR measurements (PRE) that
confirmed bending of the DNA duplex induced by the presence
of the platinum complex.
509
Using an on-column strategy, spin
label 27 (Figure 23D) was coupled to iodo-nucleotides in RNA
by the Sonogashira reaction.
510
The approach for 2-iodo-
adenine is shown in Figure 23. Nucleotide modification via
Diels−Alder reactions was also successfully investigated but not
applied for EPR or NMR spectroscopy of DNA or RNA.
511
The selective posttranscriptional labeling of RNA can be
achieved by using an expanded genetic alphabet based on the
unnatural base pair dTPT3-dNaM.
512
Subsequently, the
cyclopropene residue in the unnatural RNA nucleotide can be
selectively modified with tetrazine reagents via a [4 + 2]
cycloaddition with nitrogen release. If the tetrazine is linked to a
spin label, as in 161 (Figure 23E), this approach can be used to
introduce a paramagnetic tag into RNA after transcription from
DNA.
513
Despite the very long linker, a distance in a self-
complementary RNA duplex could be determined by DEER.
Alternatively, the dTPT3-dNAM system can be used to directly
incorporate the spin-labeled nucleotide TPT3NO into RNA by in
vitro transcription, resulting in a much shorter linker and without
the need for ligation chemistry (Figure 23E).
514
Different from proteins, lanthanoid tags for oligonucleotides
are scarce. One reason is the catalytic effect of lanthanoid ions on
hydrolytic cleavage of the phosphodiester bond if the metal ion
is not fully protected by the complexation agent. Consequently,
cyclen based tags, such as C10, are the preferred option.
296
Recently, two TAHA based lanthanide tags were reported,
which allowed the measurements of PCSs and RDCs in NMR
spectra of double-stranded DNA.
515
Both tags 68 (Figure 7) and
162 (Figure 23F) can be attached via disulfide bond formation
to a thiophenyl substituent in a selectively incorporated
nucleotide derived from EdU (Figure 23F).
3.4.5. Fully Synthetic Paramagnetic Nucleotide
Probes. Fully synthetic nucleotides are usually synthesized as
their phosphoramidite analogues for subsequent use in standard
solid-phase oligonucleotide synthesis. A summary of para-
magnetic derivatives is given in Figure 24. TEMPO-labeled
derivatives of 2′-deoxycytidine (Figure 24,163), 5-methyl-2′-
deoxycytidine (Figure 24,164), 2′- deoxyadenosine (Figure
24,165), and 2-amino-2′-deoxyadenosine (Figure 24,166)
were used in automated oligonucleotide synthesis, generating
EPR-active DNAs.
516−518
Because of the covalent link of the
TEMPO group with the base, its mobility serves as a sensitive
probe of the microenvironment, which was employed to detect
Figure 22. Convertible nucleotides for postsynthetic transformation into spin labels. The 2-fluoro-hypoxanthine derivative has also been used as O-
NPE protected version.
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single-base mismatches in duplex DNA by EPR using nucleotide
163.
519
TEMPO has also been attached via a semiflexible urea
linker in analogues 167 (Figure 24) and 168 (Figure 24).
520
The C5 position of pyrimidines is an alternative site for spin
labeling that has been studied and applied intensively (Figure
24). Using phosphotriester chemistry, TEMPO nucleotides
Figure 23. Various strategies for postsynthetic probe attachment to nucleotide bases.
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(Figure 24,169 and 170) and probe 171 (Figure 24) were
incorporated into short DNA oligomers.
521,522
Longer linkers
between pyrimidine bases and nitroxide spin labels were also
explored.
47,523,524
The first spin-labeled phosphoramidite for
incorporation into DNA was the uridine analogue 172, which
was prepared by Sonogashira cross-coupling,
525
which can be
performed during solid-phase synthesis.
526
The alkynyl linkage
limits the flexibility of this probe, making it valuable for DEER
applications to measure long-range distances with narrow
distance distributions.
527,528
The cytidine analogue 173 was
also described.
529
Sonogashira coupling yielding 172 and 173
can also be performed directly during the synthesis of
oligonucleotides,
527,530,531
similar to the attachment of 27 to
2-iodo-adenine (Figure 23).
510
Nucleotide 174
532,533
is based
on a very similar design and proven to be useful in DEER
experiments.
As an alternative, the isoindoline based spin labels ImU,OxU,
and ExlmUhave been described (Figure 24).
534,535
Their
nitroxide group is aligned with the axis of the rotatable linker
to the nucleoside, which has obvious advantages for
immobilization and distance measurements by DEER.
536
A2′-
methoxy derivative, ImUm, has also been described and used for
distance measurements in RNA duplexes.
537
Recently, the
tetraethyl nitroxide version EImUm was developed, which is
much more resistant to reduction and was, thus, successfully
used to study RNA duplexes in oocytes.
538
Hopkins and co-workers developed the nucleotide Q(Figure
24).
539,540
Pairing of Qwith the noncanonical base 2-
aminopyridine (2AP) results in a rigid spin label that has been
used to study the sequence-dependent dynamics of duplex
DNAs.
541,542
Sigurdsson and co-workers developed this concept
further by designing the rigid spin label Ç, which does not
require a noncanonical base for pairing, broadening its range of
applications (Figure 24).
543,544
Çrepresents the fusion of a
cytosine with a nitroxide featured isoindole via an oxazine
linkage. It is identical with the noncovalent spin label ç(Figure
15) except for the linkage to the ribose. Çprefers to pair with
guanine and does not perturb the structure of a DNA duplex, as
observed in a crystal structure (Figure 25).
545
A ribo-analogue
with a methoxy modification in the 2′position (Çm) has been
developed for use in RNA.
546
The synthetic incorporation of Ç
and Çminto DNA and RNA was recently improved to
circumvent partial reduction during oligonucleotide syn-
thesis.
547
Both labels deliver accurate distances in DNA and
RNA and have been used in numerous applications.
548−558
Recently, the tetraethyl nitroxide versions EÇand EÇmhave
been developed and incorporated into DNA and RNA.
559
They
are more inert toward reduction by ascorbate and thus
potentially allow in-cell EPR measurements, as recently
demonstrated for EImUm.
538
Commercially available EDTA-derivatized deoxythymidine
(EDTA-C2-dT) can be incorporated into DNA during
oligonucleotide synthesis.
560
Loaded with a Fe(II) or Mn(II)
ion, it has been used to study protein DNA interactions by NMR
measurements of PREs.
560,561
The long and flexible linker and
the occurrence of enantiomeric forms of metal−EDTA
complexes challenge applications that require the measurement
of PCSs. The design of a wider range of oligonucleotide tags that
can be used to measure PCSs is still a little explored area of
research.
Figure 24. Paramagnetic nucleotides for incorporation during oligonucleotide synthesis.
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Saxena and co-workers incorporated the Cu(II)-binding and
commercially available 2,2′-dipicolylamine (dPA) phosphor-
amidite in lieu of a nucleotide into DNA (Figure 24).
562−564
Its
use in DNA duplexes requires an abasic site in the
complementary strand and allows distance measurements by
EPR spectroscopy. A related spin label (L) was used to label
DNA quadruplexes with Cu(II) forming a Cu(pyridine)4
complex (Figure 24).
565,566
While initial studies used the
racemic form of the tag, later studies employed the (S) isomer
566
for distance measurements. A DNA quadruplex can also be
labeled with a large dinuclear platinum(II) complex linked to
TEMPO.
567
3.5. Paramagnetic Probes for Oligosaccharides
Carbohydrates present an additional class of biological macro-
molecules that have been studied with various paramagnetic
tags. To date, the variety of tags and attachment chemistries has
been more limited than for proteins and oligonucleotides.
38,39
Most strategies capitalize on the possibility of selective
amination at the reducing terminus using ammonium formate
and subsequent formation of an amide bond between tag and
oligosaccharide amine (Figure 26). Following this approach,
TEMPO has been installed at the reducing end of N-
acetyllactosamine (Figure 26A, 175) to study its interaction
with proteins by NMR spectroscopy.
568,569
Subsequently, a
more complex high-mannose-type oligosaccharide was studied
with the same approach.
570
An EDTA-based lanthanide tag was first attached to the
aminated reducing terminus of N,N′-diacetylchitobiose (Figure
26B, 176) to enable paramagnetic NMR measurements with an
excellent correlation between experimental and predicted
PCSs.
571
Subsequently, this tagging strategy was also applied
to high-mannose-type oligosaccharides.
572,573
Biphenyl variants
Figure 25. X-ray crystal structure (PDB code 3OT0)
545
of a 10 base
pair DNA duplex containing the Çspin label (highlighted in pink). Two
uridine residues are 2′-methylated. The phosphate backbone is shown
as a ribbon. The distance between both nitroxide radicals (18 Å) is
indicated. Hydrogen bonds between guanine and Çare indicated by
dashed lines.
Figure 26. Carbohydrate probes and their synthesis.
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of this tag in (S)(Figure 26B, 177)
574
and (R)(Figure 26B,
178)
575
configurations were also described.
Kato and co-workers developed a phenylenediamine based
lanthanide chelating tag that can be coupled to the aminated
reducing terminus of oligosaccharides using standard amide
bond formation (i.e., HATU (2-(7-azabenzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium hexafluorophosphate), DIPEA
(N,N-diisopropylethylamine), and DMF (N,N-dimethylforma-
mide)) yielding 179 (Figure 26B).
576
This tag has since been
used in various applications to study carbohydrates and their
interactions with proteins by paramagnetic NMR spectrosco-
py.
577−580
A biphenyl variant attached to lactose (Figure 26B,
180) has also been used to study lactose conformations and its
interactions with a receptor protein.
581
Another report described the use of an L-fucose azide and an
alkyne based tag for direct “click”reaction to furnish compound
181 (Figure 26C).
582
It provided an elegant way to obtain a
model for the binding site of the fucose on a 70 kDa receptor
protein by the use of intermolecular PREs and PCSs to position
the lanthanoid ion on the carbohydrate with respect to the
protein. In a partially enzymatic and partially synthetic approach,
an azide bearing saccharide was selectively introduced to a
glycosylation site in a protein and subsequently reacted with an
alkyne based label to yield the spin-labeled glycoprotein 182
(Figure 26D).
583
4. COMPLICATIONS IN PRE-TO-DISTANCE
CONVERSION
The PRE is very strong for a nucleus Iclose to a paramagnetic
center Sand drops offrapidly with increasing distance rIS
because of its rIS−6dependence (eqs 9−13). Therefore, PREs
are powerful tools for the detection of minor states or the
characterization of ensembles of conformations, such as found in
intrinsically disordered proteins or encounter complexes.
33,34,584
At the same time, the strong distance dependence of the PRE
adds uncertainties to distance measurements of major
conformations, when minor conformations or alternative
interactions featuring short distances rIS contribute dispropor-
tionally to the observed PRE. Minor species populated as low as
0.1% can be manifested in the observed PRE in this way.
Most obviously, problems arise when proteins are prone to
aggregation, but the potential for intermolecular associations
may also be introduced by the chemical tag. In principle,
intermolecular effects can be detected by measuring the PREs at
different concentrations of the tagged molecule,
365
but accurate
measurements of PREs at low concentration may be very time-
consuming for biological macromolecules. Intermolecular PREs
can be suppressed by dilution of the paramagnetically labeled
molecule with the diamagnetic reference, but as the chemical
shifts are conserved in the presence of paramagnetic centers with
slow electronic relaxation times, the superimposition of NMR
signals from diamagnetic and paramagnetic molecules makes it
difficult to disentangle the intra- and intermolecular PREs.
A second complication arises from incomplete paramagnetic
tagging. Tags with limited metal binding affinity are easily under-
or overtitrated with metal ions and nitroxide tags may be subject
to partial reduction or oxidation to diamagnetic species, leading
to mixtures of paramagnetic and diamagnetic species even
following quantitative chemical ligation. These complications
may explain, why the correlation of PREs with distance has been
disappointing in many published examples, despite the steep
distance dependence of the PREs, compromising the accurate
conversion of PREs into distances.
130,365,434,459,461,585−587
Finally, the PRE-to-distance conversion is compromised by
any flexibility of the paramagnetic tag as well as uncertainties in
electronic relaxation rates. In the case of tags containing Gd(III),
Mn(II), or Cu(II) ions as the paramagnetic center, the PRE is
entirely or predominantly driven by the SBM mechanism, which
depends on the effective correlation time of the vector
connecting the nuclear spin with the paramagnetic center. In
addition, the electronic relaxation rate depends on the ligand-
field of the metal complex. In practice, conversion of the PREs
into distances, thus, requires calibration against known distances
to atoms in the target molecule.
249,588
In the case of nitroxide
tags, the electronic relaxation time is very long (of the order of
100 ns),
589
but the generally hydrophobic nature of these tags
makes the assumption of absence of intra- and intermolecular
associations more tenuous.
The problem of incomplete paramagnetic tagging and
uncontrolled intermolecular PREs can be solved by using
metal tags with anisotropic magnetic susceptibility tensor. In this
case, the NMR signals of paramagnetic and diamagnetic species
are separated by PCSs. Furthermore, intermolecular PREs can
readily be accounted for by mixing the paramagnetically tagged
molecule with the diamagnetic reference. In the mixture, the
relaxation rates of diamagnetic and paramagnetic species are
equally affected by intermolecular PREs, so that the
intermolecular effects drop out when calculating the difference
in relaxation rates between paramagnetic and diamagnetic
species. The concept was explored and demonstrated with
calbindin D9k containing Er(III) or other paramagnetic
lanthanoid ions as the paramagnetic center and Lu(III) as the
diamagnetic reference.
124
The results showed that PREs
extracted from longitudinal relaxation rates were less sensitive
to intermolecular effects than transverse PREs but that more
accurate distance restraints were obtained by measuring
transverse relaxation rates as measurements of R1(1H) relaxation
rates are affected by multiexponential relaxation due to cross-
relaxation. Specifically, transverse PREs measured with the
Er(III) sample delivered reliable distances in the range between
12 and 25 Å with an RMSD below 1 Å, if RDCs in the
paramagnetic state were controlled by using short relaxation
delays and a magnetic field strength not above 14.1 T. In the case
of flexible tags, however, distance variations and difficulties to
determine the effective correlation time remain compromising
factors in quantitative PRE-to-distance conversions.
5. EXAMPLES OF APPLICATIONS OF PARAMAGNETIC
NMR IN PROTEIN STUDIES
Paramagnetic effects provide structural information, which can
be used further to assess the dynamics of biological macro-
molecules, such as domain reorientation or the exchange with
minor conformational species. The following section serves to
give examples that illustrate the range of applications of
paramagnetic probes, including protein resonance assignment,
3D structure determination, studies of protein−protein and
protein−ligand complexes, and the identification of lowly
populated states. Section 6 adds further examples for an
overview of the breadth of applications for the different types of
probes.
5.1. Protein Structure Studies
5.1.1. NMR Resonance Assignments in the Para-
magnetic and Diamagnetic States. Sequence-specific
resonance assignments are the starting point for any protein
structural study by NMR spectroscopy. If the protein is not too
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large and amenable to uniform labeling with 15N, 13C, and 2H,
the resonance assignment generally is easiest by heteronuclear
multidimensional (3D/4D) NMR experiments. For large
proteins, complete resonance assignments become challenging
because of increasing line width and spectral overlap. PCSs offer
a way to increase the spectral dispersion and thus resolve spectral
overlap. This is particularly useful for correlation spectra of
methyl groups, which otherwise show limited spectral
resolution.
358
If the 3D structure of the target protein is known in advance,
PCSs generated by a site-specifically attached probe can be used
to assist the resonance assignments.
590
Fundamentally, measur-
ing PCSs requires attributing the chemical shift observed for a
nucleus in the paramagnetic state to its chemical shift in the
diamagnetic state. Different methods have been devised to
achieve this.
591
Most commonly, 2D correlation spectra, such as
heteronuclear single-quantum correlation (HSQC) spectra, are
used, where the cross-peaks of paramagnetic and diamagnetic
samples are displaced along approximately parallel lines. This
observation holds true in particular for paramagnetic metal ions
featuring large Δχtensors as, in this case, PREs broaden the
signals of nuclear spins near the metal ion beyond detection and
PCSs can be observed only for nuclei beyond a minimal distance
from the paramagnetic center. As the distance between two
adjacent nuclear spins is much shorter than the distance from the
paramagnetic center, very similar PCSs are observed for both
spins involved in a cross-peak in 2D correlation spectra or
spectra of higher dimensionality, leading to the cross-peak
displacements along approximately parallel lines. This greatly
facilitates assigning the paramagnetic peaks to their parents in
the spectrum recorded of the diamagnetic reference and the
attribution can further be underpinned by using two para-
magnetic samples produced with the same probe but different
paramagnetic metal ions, which generate PCSs of different
magnitude. In this case, all three peaks (two paramagnetic, one
diamagnetic) produced between a pair of nuclear spins can be
expected to lie on the same line. It is further helpful that, for
many probes, the Δχtensors of Tb(III) and Tm(III) ions tend
to shift the cross-peaks in opposite directions relative to the
diamagnetic reference (Table 2).
290,158,258,265
Because the PCSs
differ for different protons in a molecule, spectral overlap makes
it very difficult to measure proton PCSs by 1D NMR
spectroscopy.
In an unbiased way, the assignment of paramagnetic peaks to
their diamagnetic parent can be achieved by a spectrum that
generates cross-peaks between both. This requires a sample
simultaneously containing both the paramagnetic and diamag-
netic species, where the probes installed bind the metal ions in a
way that allows chemical exchange between paramagnetic and
diamagnetic metal ions in the slow-exchange regime (exchange
rate between about 1 and 100 s−1). These stringent criteria have
been fulfilled in some examples, such as probes 125
327
and
126,
328
but are difficult to design in advance.
330,592,593
Notably,
the situation of fast chemical exchange also provides a
straightforward way of measuring PCSs as, in this case, they
increase continuously with increasing concentration of the
paramagnetic metal probe.
365,591
Lesser binding affinity,
however, comes with the risk of lesser binding specificity,
which can make it difficult to interpret the PCSs.
Having measured a sufficient number of PCSs to fitaΔχ
tensor to the 3D structure of the biological macromolecule, the
Δχtensor can be used to predict further PCSs to assist with the
resonance assignment. When using a double-armed probe like
CLaNP-5 (Figure 9,79) that is relatively rigid relative to the
protein, it is also possible to predict an initial Δχtensor with
good accuracy.
127
As nuclei located at different positions relative
to the paramagnetic center can have the same PCS, the
distinction needs to be made by additional data, such as PREs,
RDCs, and CCR effects,
57
or by PCSs generated from more
paramagnetic centers installed at different sites of the protein.
The software packages PLATYPUS,
57
Echidna,
594
Possum,
595
and PARAssign
596
were developed specifically for resonance
assignment using paramagnetic restraints.
An instructive example is the assignment of the diamagnetic
resonances of methyl groups of the N-terminal domain of Hsp90
with CLaNP-5 tags (Figure 9,79) installed at three different
sites. The protein contains 76 methyl groups in leucine,
isoleucine, and valine residues, and over 60% of them could be
assigned based on the protein structure and 1H PCSs measured
in 13C-HSQC spectra with Yb(III) as the paramagnetic metal
ion, using PARAssign without the use of any prior assignments
from the diamagnetic sample.
597
For the remaining methyl
groups the choice of possible assignments was greatly reduced.
As the Δχtensor orientations of the S50C/D54C and T149C/
I187C mutants happened to be almost parallel, the information
content was greatly reduced and the assignment was
predominantly based on two Δχtensors only. If the Δχtensor
has been determined independently, for example, from 15N-
HSQC spectra of backbone amides, PCSs from a single site with
a single paramagnetic metal ion can be sufficient to assign 75% of
the methyl resonances in the diamagnetic state using the
program Possum, as shown for the 30 kDa complex between the
N-terminal exonuclease domain ε186 and the subunit θof the E.
coli DNA polymerase III (ε186/θ).
595
By starting from a X-ray
crystal structure, the work also highlighted the fundamental
problem of this approach, which arises when subtle differences
exist between crystal and solution structures.
5.1.2. Protein Structure Determination. PCSs arguably
deliver the most useful paramagnetic structure restraints for 3D
structure determinations of proteins, because they are readily
measured with high accuracy. In addition, RDCs obtained by
paramagnetic alignment convey useful information about
relative domain orientations. Paramagnetically generated
molecular alignment and measurement of RDCs also is of
interest for studies of protein dynamics,
598,599
which rely on
multiple different alignment tensors, as it can be challenging to
obtain a larger number of significantly different alignments with
the help of conventional alignment media, which act mostly by
steric or electrostatic effects.
600
Software packages like
CYANA,
601
Xplor-NIH,
70,602
and Rosetta
603−607
have been
extended with modules for protein structure computation with
paramagnetic restraints.
In the 1990s, Bertini and co-workers refined 3D structures of
proteins with the help of paramagnetic data, demonstrating that
the structural restraints gained by paramagnetism can outweigh
the loss of NMR signals from 1H spins in the vicinity of the
paramagnetic center.
112,139
More recently, the benefitof
paramagnetic data for 3D structure determinations was
highlighted by Nietlispach and co-workers, who applied PCS
restraints to refine the structure of the phototactic receptor
sensory rhodopsin II (pSRII), a membrane protein with over
240 residues from the family of seven transmembrane helix
proteins.
608
In previous work, the authors had determined the
structure of pSRII in a micelle using NOEs.
609
By labeling pSRII
at four different positions with the paramagnetic probe C2
(Figure 9,96) loaded with different paramagnetic metal ions,
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the global fold of the protein could be derived by the exclusive
use of PCS restraints. By combining the PCS and NOE data, the
structure was refined to a backbone RMSD of 1.5 Å to the
mean.
608
Besides delivering unique long-range structural restraints, the
chemical shift measurements needed to measure PCSs can be
performed very quickly. This has recently been exploited to
elucidate the structure of the transient enzyme intermediate
formed by SrtA with its substrate peptide.
266
The enzyme−
substrate complex is an unstable intermediate that contains a
labile thioester bond with the active-site cysteine residue
(Cys184) and presents only a minor species in solution.
Nonetheless, comparison with the chemical shifts of a stable
disulfide-bond-linked analogue enabled the resonance assign-
ment of the thioester intermediate and attachment of the probes
76 and 77 (Figure 7) loaded with one of four lanthanoids,
Dy(III), Tb(III), Tm(III), and Lu(III) to SrtA delivered PCSs.
Using the PCSs as input for Xplor-NIH, the structure of the
thioester intermediate was calculated as an ensemble of
conformers with a RMSD of 1.1 Å.
266
The sensitivity with which PCSs can be measured is also of
great advantage for measurements at low concentrations, such as
encountered in in-cell studies of protein structure. Using GB1 as
a model protein, Müntener et al. attached the M7Py-DOTA tag
(Figure 9,88) loaded with one of three lanthanoids, Tb(III),
Tm(III), and Lu(III), and recorded PCSs and RDCs at about 50
μM protein concentration after transferring the labeled protein
into Xenopus laevis oocytes.
286
The paramagnetic restraints
measured by in-cell NMR assisted the 3D structure determi-
nation using GPS-Rosetta.
606
The same protein at the same low
concentration was targeted at the same time by Pan et al., using
PCSs measured in X. laevis oocytes.
610
To measure PCSs, this
group attached 4PS-PyMTA (Figure 7,76)loadedwith
Tb(III), Tm(III), Yb(III), or Y(III) ions and calculated the
structure without RDCs using GPS-Rosetta. The authors were
careful to point out, however, that Rosetta
611
is one of the most
powerful software packages for 3D structure predictions of small
proteins from the amino acid sequence alone.
610
Specifically,
they showed that the fold of GB1 is already correctly predicted
by CS-Rosetta,
603
when backbone chemical shifts are provided
as the only experimental data.
610
The conversion of PCS data into structure restraints requires
knowledge of the Δχtensor, which is best obtained by fitting to
3D structure coordinates. For proteins of unknown structure,
the powerful model building algorithm provided by Rosetta thus
is an excellent starting point where models that do not allow
good Δχtensor fits can be discarded early in the structure
calculation.
603
In this way, PCS data not only improve the
quality of the final structures but also accelerate the convergence
of the calculations. The value of PCS restraints for protein fold
determination, even if backbone chemical shifts and PCSs from
backbone amide protons are the only experimental data
provided, is very clear for larger proteins.
604,605,607,612
Because of their long-range nature, PCSs are particularly
suited to determine full structures in situations, where the
structure of a rigid protein domain is known while the
conformation or orientation of another part is not. In this
situation, even sparse PCS data have been shown to be sufficient
to fitΔχtensors to the rigid part and use the PCSs of the
unknown part to determine its structure or relative orienta-
tion.
147,291,612−619
It is instructive to compare the PCS structure restraints with
the way, in which the global positioning system (GPS) identifies
the location of objects on Earth. Just as the measurement of the
distances to several satellites identifies a single location on the
surface of Earth, the PCSs of a nuclear spin can be measured for
different protein samples prepared with tags positioned at
different sites. Each PCS defines an isosurface and the
intersection of four PCS isosurfaces defines a single point in
space.
606,620
The concept was demonstrated with the 3D
structure determination of the C-terminal domain of ERp29
tagged at four different sites either with IDA-SH (Figure 11,
126)orC1 tags (Figure 9,96), using Tm(III) and Tb(III) as the
paramagnetic ions.
606
The same concept was applied to
determine the structure of this protein with the help of PCSs
measured with Co(II) ions bound to double-histidine motives in
four different helices, one at a time.
592
Notably, PCSs of the
backbone amide protons alone were sufficient to obtain the
correct protein fold. While PCSs from an increasing number of
tagging sites increases the level of structural information
available, structural details on the conformations of amino
acid side chains can already be obtained with two different
tagging sites, as the side chain locations are restricted by their
link to the backbone.
94,621
5.1.3. Protein−Protein Interactions. With a paramagnetic
probe on one of the interacting proteins in a protein−protein
complex, the paramagnetic effects in the partner protein can be
recorded to determine the relative position and orientation of
the two proteins in a rigid body docking approach, which
assumes that the structures of the individual binding partners are
conserved in the complex (Figure 27). The first example of this
approach, from 1998, used the paramagnetic heme in
cytochrome fto induce PCS in the electron transfer partner
plastocyanin and model the structure of the complex.
622−624
In
an early example using lanthanoid ions, the structure of the
ε186/θcomplex was modeled with the help of PCSs measured
with Dy(III), Ho(III), or Er(III) data in the natural metal
binding site of ε186, using a rigid body docking approach,
although the PCSs were also used to refine the structure of θin
its bound state.
69,625
PCSs were also successfully used to model the structure of a
homodimer. In this case, two mutants of p62 PB1, named DR
and KE, were produced to prevent oligomerization beyond
dimer formation.
159
A two-point anchored LBP was incorpo-
rated at the N-terminus of DR to coordinate with paramagnetic
lanthanoid ions. The lanthanoid position relative to the DR
mutant was determined by fitting the Δχtensors and PCSs were
measured in the KE mutant. Using the measured PCS and
chemical shift changes observed at the interface of the KE
mutant, rigid body minimization protocol in Xplor-NIH
70,602
delivered the structure of the dimer. It was noted, however, that
multiple solutions were possible even with the availability of
PCS data sets from different lanthanoid ions as the Δχtensor
axes tended to be similar. The degeneracy could be resolved by
modifying the LBP.
626
Protein−protein interactions (PPI) are based on noncovalent
forces, such as charge−charge attractions, hydrophobic
interactions, and hydrogen bonds, and paramagnetic effects
are excellent tools to investigate very weak protein−protein
interactions such as observed in encounter complexes. As the
protein partners approach each other in solution, the formation
of an encounter complex precedes the formation of the specific
protein−protein complex. These weak and dynamic complexes
are essential for rapid complex formation, yet difficult to
characterize by traditional techniques.
627,628
PREs are partic-
ularly suited for investigating such weak PPIs, as they are
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insensitive to the relative orientation of the proteins and
sensitive to minor states in which a nucleus is closer to a
paramagnetic center than in the major state, which usually is the
final specific complex. Complementary information can be
obtained by PCSs and RDCs, provided that the orientation of
the Δχtensor changes only over a limited angular range, when
the proteins populate an ensemble of complex structures.
Averaging over a greater range is evidenced by averaging of the
PCSs and RDCs to zero.
The electron-transfer complex of yeast iso-1-cytochrome c
(Cc) and yeast cytochrome cperoxidase (CcP) is an example of a
weak PPI comprehensively characterized by paramagnetic
NMR. Both proteins contain a paramagnetic heme group, but
the intermolecular PCSs are too small to be suitable for
structural studies. Following the attachment of a paramagnetic
tag (MTSL,Figure 3)atdifferent locations on the CcP surface,
PREs were recorded for Cc and utilized to dock Cc on CcP. The
resulting model of the complex was similar to the crystal
structure. In this case, the encounter complex represented no
less than 30% of the complex and its interface was preferentially
located near that of the final specific complex.
65,91
CcP variants
with added negative patches were used to study the role of
charge distribution and PREs showed that cytochrome cvisits
such new patches in the encounter complex.
83
In another example, the complex between cytochrome
P450cam (cytP450cam) and putidaredoxin (Pdx) was inves-
tigated using CLaNP-7 (Figure 9,80) loaded with Tm(III) or
Gd(III) to obtain intermolecular PCSs, RDCs, and PREs.
629
The position of putidaredoxin on cytP450cam found with these
restraints was in good agreement with crystal structures
obtained independently.
629,630
The study highlighted the value
in combining different types of paramagnetic information to
obtain a good structure. In addition, the NMR data provided
evidence for the presence of a lowly populated encounter
complex. Using additional paramagnetic restraints and the
maximum occurrence of regions methodology,
631
it could be
shown that in this encounter complex Pdx visits a large area of
the surface of cytP450cam.
With increasing molecular weight of the system, it becomes
increasingly difficult to obtain the specific NMR resonance
assignments required to fitΔχtensors. In this situation, site-
specific incorporation of a probe that can easily be observed in
the NMR spectrum can be combined with a paramagnetic probe
to monitor the PRE on the NMR probe and, thus, derive
distance restraints. The concept has recently been demonstrated
by Abdelkader et al., who used genetic encoding for site-specific
incorporation of an unnatural amino acid carrying a
trimethylsilyl (TMS) group at the end of the amino acid side
chain to obtain a readily observable 1H NMR probe (an intense
singlet near 0 ppm).
632
Using a 71 kDa homodimeric p-cyano-L-
phenylalanyl-tRNA synthetase as an example, a mixture of TMS
labeled protein and paramagnetically labeled protein (using
either MTSL or the C1 tag loaded with Gd(III) attached to a
cysteine residue) revealed a PRE across the dimer interface with
Gd(III) but not with MTSL.
632
Taking into account the
flexibilities of the tags and TMS amino acid side chain, the data
could be interpreted as a distance restraint between about 10
and 20 Å.
5.1.4. Protein−Ligand Interactions. NMR spectroscopy
is uniquely suited to study protein−ligand interactions in drug
discovery campaigns because NMR works in solution and can
provide atomic level information about the binding site and
ligand orientation. It is useful to distinguish between the
scenarios where the exchange rate of the ligand is fast or slow on
the time scale of the NMR experiment. Rapid and slow ligand
binding and dissociation generally correlates with relatively
weak and strong ligand affinities, respectively. Paramagnetic
NMR opens new approaches for studies in either limit.
Fragment-based ligand screening is a method that searches for
small molecules that bind to proteins weakly. The use of
paramagnetic NMR for fragment-based ligand screening has
been explored by Saio et al. with a double-anchored LBP.
633
Binding of the ligand was reported by PREs generated by a
Gd(III) ion in the LBP, where the PREs were detected as
increased transverse relaxation rates of the 1H signals in 1D
NMR spectra of the ligand, using T1ρrelaxation experiments. In
the limit where the ligand exchanges between free and bound
state faster than the transverse relaxation rate, the relaxation
enhancement experienced in the bound state due to the
proximity of the paramagnetic center in the target protein is
transferred to the spectrum of the free ligand. In analogy to the
transferred NOEs and saturation transfer, the effect is referred to
as transferred PRE (tPRE). The size of a tPRE reflects the
distance between the nuclear spin of the ligand in the bound
state and the paramagnetic center attached to the target protein.
Figure 27. Strategy for using PREs, PCSs, and RDCs for structure
determination of protein−protein complexes using differently labeled
samples. PREs are measured using a tag with slow electronic relaxation
(e.g., Gd(III)) or a paramagnetic center with little χtensor anisotropy
(e.g., Er(III)). PCSs and RDCs are obtained using the tag with an
anisotropic paramagnetic center (e.g., Tm(III)). The data are measured
with respect to a diamagnetic reference produced with the tag
containing, for example, a Lu(III) ion. To simplify the NMR spectra
and distinguish between intermolecular and intramolecular effects, the
complex can be prepared with isotope labeling (symbolized by green
dots) of one of the proteins only. The intramolecular restraints are used
to fit the position of the paramagnetic center and orient the Δχtensor.
2D NMR spectra (typically [15N,1H]-HSQC spectra) can be sufficient
to obtain a complete set of inter- and intramolecular PREs, RDCs, and
PCSs, which allow determining the relative position and orientation of
the two proteins in a rigid body docking approach. Reproduced with
permission from ref 30. Copyright 2014 Elsevier.
Chemical Reviews pubs.acs.org/CR Review
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Chem. Rev. XXXX, XXX, XXX−XXX
AP

In the same way, also transferred PCSs (tPCS) can be observed
and used to assess the structure of protein−ligand complexes.
633
In another example, Guan et al. demonstrated that the
location and orientation of a ligand can be determined solely
using tPCSs, provided that the ligand is in fast exchange between
bound and free states relative to the size of the PCS (expressed in
rad s−1).
620
In the fast exchange limit, the experiments allow
using the free ligand in excess, that is, only a small fraction of the
ligand is bound and the PCSs measured are scaled by the bound
fraction. Although the PCSs measured are thus relatively small,
the smaller size is more than compensated by the narrow line
width observed for the ligand, which is dominated by the
fraction of free ligand. Placing the paramagnetic center, Ln(III)-
CLaNP-5 (Figure 9,79), at three positions, one at a time, on the
target protein FKBP12 (Figure 28) enabled a PCS-based
triangulation approach to capture the ligand’s site of binding and
orientation relative to the protein. The structure of the complex
obtained from tPCSs agreed with the restraints obtained from
NOEs but was easier to obtain in the absence of extensive 1H
NMR resonance assignments of the protein.
In an early example, John et al. showed that tPCSs generated
by a single lanthanide binding site can be sufficient to determine
the binding site and orientation of a weakly bound ligand, if
steric restrictions imposed by the protein invalidate any of the
alternative structural solutions of the tPCS data set.
634
In this
example, the lanthanide ion was bound deep inside the metal
binding pocket of the ε186/θcomplex. The ligand (thymidine)
bound with millimolar binding affinity and tPCSs were
observable in 1D 1H and 13C NMR spectra of the ligand present
in over 500-fold excess over the protein complex. The sensitivity
of the approach is illustrated by the speed (<0.5 h) with which
the 13C NMR spectra were recorded at natural isotopic
abundance. The binding mode coincided closely with that
observed in the crystal structure of the ε186−thymidine
monophosphate complex.
A caveat associated with tPRE and tPCS effects is the
possibility that the weakly binding ligand temporarily associates
with the paramagnetic tag rather than the target molecule. False
positives arising in this way cannot adequately be addressed by a
control experiment probing the direct interaction between
ligand and free paramagnetic tag, as unexpected interactions are
likely promoted by the formation of binding pockets between
tag and target molecule. The strong distance dependence of
PCSs and PREs notably amplifies the effects of minor
populations of direct ligand−tag interactions.
As ligand binding tends to affect the global structure of the
target molecule little or not at all, changes in PCSs observed
locally in the target molecule can be used to determine local
structural changes arising from the interaction with a ligand,
such as changes in amino acid side chains. A recent study labeled
the protein FKBP12 with Ln(III)-CLaNP-5 (Figure 9,79)at
three sites, and valine and leucine residues were selectively
isotope labeled with 13CH3groups.
621
Significant changes in
PCSs were observed between the free and ligand-bound protein
for methyl groups located near the ligand binding site, indicating
displacements of the methyl groups in the 1−5 Å range.
In the slow exchange limit, it is not meaningful to use ligand in
excess over the target protein, which makes it much harder to
discriminate the NMR signals of the ligand against the
background of protein signals, especially as the chemical shifts
of the bound ligand usually differ from those in the free state.
While isotope labeling of the ligand enables its selective
detection by NMR spectroscopy, the chemical synthesis of
isotope labeled ligands is cumbersome in practice. Chen et al.
demonstrated that chemical labeling of the ligand with a tert-
butyl group presents a synthetically more practical alter-
native.
635
As the 1H NMR signal of the tert-butyl group is an
intense signal, it can readily be detected by 1D 1H NMR. The
specific example used was the 27 kDa complex between the
dengue virus NS2B-NS3 protease, tagged at three different sites
with C2 tags loaded with Tm(III), Tb(III), or Y(III), and a
covalently binding peptide inhibitor labeled with a tert-butyl
group.
635
NOESY spectra revealed PCSs not only of the tert-
butyl group but also of ligand resonances of protons near the
tert-butyl group, which enabled positioning and orienting the
ligand in the bound state. Although the structural information
obtained for the ligand in this way appears limited, it proved
decisive in identifying the correct pose obtained by independent
modeling of the protein−ligand complex.
5.1.5. Structures of Minor States. Protein folding, enzyme
catalysis, and other functions require conformational exchange,
involving excited states with low populations and short lifetimes
(microseconds to milliseconds). The structures of these lowly
populated states can usually not be solved by X-ray
Figure 28. Transferred PCSs identify the binding site and binding orientation of ligand molecules that are in fast exchange with excess free ligand. (A)
tPCSs (the difference between the resonance positions for the paramagnetic (para) and diamagnetic (dia) samples) observed in the 1H 1D NMR
spectra of excess ligand in complex with the protein, which is tagged with either paramagnetic or diamagnetic ions (one site at a time). The dashed and
solid lines identify the ligand resonances in the diamagnetic and paramagnetic samples, respectively. (B) Superposition of the averaged NOE structure
(in green) of the FKBP12−1 complex and the best five structures (in orange) calculated using tPCSs. The protein backbone is represented as a gray
ribbon except for the residues Asp37 and Tyr82 (yellow). The average RMSD of the ligand from PCS calculations relative to the NOE calculation is 2.8
±0.4 Å. Reproduced with permission from ref 620. Copyright 2013. American Chemical Society.
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AQ

Table 4. Applications of Synthetic Probes in Protein NMR Studies
probe applications in protein NMR studies comments
MTSL (Figure 3) protein global folding and structure
determination
130,586,649,652,656,668,669,671
small tag generating sizable PRE effects; easy to handle and commercially available; in NMR experiments, this tag produces PREs as the only
paramagnetic effect; the nitroxide group is sensitive to reduction or oxidation; reduction of the disulfide bond formed with the target leads to loss of
the tag
protein−protein interaction
65,83,91,658
protein−ligand interaction
661−663
protein dynamic studies
664−667,670,730
protein distance measurements by 19F-
PRE
587
maleimide-TEMPO
(Figure 5,15)protein conformation studies
667
commercially available and attached to protein via stable thioether linker
SPy-EDTA and analogues
(Figure 7,60−61)protein structure
determination
249,250,643,644,674,675
commercially available, but exists in multiple conformations
protein binding target determination
676
protein alignment
251
4MMPyMTA and
analogues (Figure 7,
73−77)
protein structure determination
266
and in-
cell protein structure determination
610
lanthanoid based probe for attachment to single cysteine residues; small, hydrophilic, and carrying a low net charge; forming a stable thioether linkage
with cysteine; complete ligation yields can be difficult to obtain
protein−peptide complex
263
protein−ligand interactions
678,679
protein complex NMR signal
assignment
677
CLaNP-5 (Figure 9,79) methyl group assignment
597,621
frequently used lanthanoid-based cyclen probe, suitable for PCS measurements; the double-armed linkage to the target results in high rigidity; it carries
a net charge of 3+ and some of its pendants are fairly hydrophobic; the disulfide linkage between probe and target is prone to reduction
protein−ligand interaction
studies
76,620,617,641
protein structure determination
616,682,731
protein dynamic studies
274,275
protein−protein complex studies
680
protein−peptide complex studies
681
CLaNP-7 (Figure 9,80) protein complex studies
629,631,685−687
similar to CLaNP-5, but less charged and with yellow color
protein structure determination
629
protein conformation studies
685
DOTA-M8 (Figure 9,
86)intrinsically disordered proteins; increasing
NMR spectral dispersion
694
binds Ln(III) tightly, produces largeΔχtensors, and has zero net charge; disulfide bond after attachment to the protein may be prone to reduction; two
conformations were reported.
protein domain studies
695,696
protein alignment
697
protein−ligand interaction
698
RNA structure analysis
699
protein structure refinement
701
M8-CAM-I (Figure 9,
87)in-cell protein structure determination
285
shares the core structure with DOTA-M8 but a propyliodoacetamide attachment replaces the pyridylthio linker to form a more stable C−S linkage; a
long tether to the protein backbone reduces its Δχtensor magnitudes
M7Py-DOTA (Figure 9,
88)in-cell protein structures from 2D NMR
experiments
286
an analogue of DOTA-M8, with a 4-(phenylsulfonyl)pyridine moiety yielding attachment with a short and rigid linker but the attachment reactivity is
low
M7PyThiazol-DOTA
(Figure 9,92)homotrimeric protein structure
investigation
700
a newer version of M7Py-DOTA; reacts with cysteine with higher selectivity and efficiency; generates a Δχtensor even larger than DOTA-M8
protein−ligand interaction
698
protein structure refinement
701
C1/C2 (Figure 9,96) methyl group assignment
689
lanthanoid based cyclen probe for attachment to single cysteine residues; suitable for PCS measurements but with high net charge and bulky
hydrophobic pendants; forming a disulfide linkage between probe and target; C1 and C2 are enantiomers
protein structure determination
94,604,606,612
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AR

Table 4. continued
probe applications in protein NMR studies comments
protein−protein interactions and protein−
ligand interactions
291,618,619,690
DNA−protein domain binding studies
693
C3 (Figure 9,98) protein structure studies
702
similar to the C1 and C2 tags but for attachment to p-azido-L-phenylalanine by Cu(I)-catalyzed click chemistry, which is orthogonal to cysteine
chemistry
TEMPOL (Figure 12) protein accessible surface
characterizations
345,346,355,711−714,732,733
commercially available and frequently used but somewhat hydrophobic; unstable under reducing conditions
BPTI aggregation
715
protein folding
347
protein dynamics studies
664−667,670,730
protein distance measurements by19F-
PRE
587
Gd(III)DTPA-BMA
(Figure 13)protein surface accessible regions
characterizations
350,716,734
high solubility, stability, and low protein binding affinity; zero net charge
BPTI aggregation
715
protein structure determination
352,721
protein complexes studies
719,723
protein domain studies
722
spectral editing
726
Gd(III)TTHA-TMA
(Figure 13)protein dynamic determinations
353
zero net charge
[Ln(III)(DOTP)]5−
(Figure 13)protein−protein interaction
718
high negative charge; can generate both PCSs (loaded with Tb(III)) and PREs (loaded with Gd(III)) in some cases
[Ln(III)(DPA)3]3−
(Figure 13)multisite protein−ligand binding
interactions
724
can generate both PCSs (loaded with Tb(III)) and PREs (loaded with Gd(III)) in some cases
cationic peptides structure
determination
725
[Ln(III)(DTPA)]2−
(Figure 13)spectral editing
358
negatively charged
Gd(III)(DO3A) (Figure
13)identification of residues close to
carboxylates
366
commercially available and zero net charged
[Ln(III)(EDTA)]1−
(Figure 13)protein−protein interface mapping
357
commercially available and negatively charged
spectral editing
358
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AS

crystallography. Relaxation dispersion measurements using the
Carr−Purcell−Meiboom−Gill (CPMG) sequence and T1ρ
experiments, as well as chemical exchange saturation transfer
(CEST) experiments are NMR methods, which have proven
uniquely suitable for the study of such high-energy states of
proteins.
636−638
Relaxation dispersion experiments can yield the absolute
difference in chemical shifts (Δδ) of nuclear spins between the
minor and major states. In combination with RDC restraints
obtained in an external alignment medium, the structure of a
minor state can be determined in favorable cases.
639,640
It is still
difficult, however, to convert the limited experimental
information into a 3D structure. In this situation, PCSs of the
minor state would provide powerful structural restraints. If the
PCSs of the minor state differ from those of the major state, they
can be extracted from a relaxation dispersion experiment, where
the diamagnetic control sample delivers the chemical shift
change in the diamagnetic state and the paramagnetic sample
delivers the Δδvalues including the PCSs.
275
For this
application, rigidity of the paramagnetic center is critically
important to obtain accurate structural restraints. Earlier
relaxation dispersion studies of cardiac troponin C substituted
with lanthanoids (Ce(III) or Pr(III)) indicated that the PCSs
observed most likely reflect motions of the paramagnetic
center.
641
To avoid this problem, both the attachment site and
the probe must be rigid on the millisecond time scale. When
CLaNP-5 loaded with Tm(III), Yb(III), or Lu(III) was linked
to Paz and Cc, which are known to be rigid proteins, 1H CPMG
relaxation dispersion experiments revealed dispersion effects
likely reflecting movements of the tag rather than protein.
275
When CLaNP-5 was attached to adenylate kinase, relaxation
dispersion experiments similarly indicated the presence of a
minor conformational state of the probe, in which the Δχtensor
is rotated by 20°relative to the major state. This work illustrated
the difficulty of finding a probe without any exchange
phenomena on the millisecond time scale. In particular, ring
flips of the cyclen ring are hard to suppress.
274
Alternatively, PCS restraints for determining the structure of a
minor state can be derived from CEST experiments. For a proof
of concept of PCS-CEST experiments, Ma et al. used Abp1p
SH3−Ark1p, which is a protein−peptide complex with a slow
off-rate of the bound peptide, as a model system.
263
4MMPyMTA (Figure 7,73) loaded with Tb(III) was attached
to the SH3 domain and the minor state was produced by adding
only 0.03 equiv of Ark1p peptide. The 15N PCSs of the minor
species determined by the CEST measurements proved to be in
agreement with the 1:1 Abp1p SH3−Ark1p complex. The same
paramagnetic tag was attached to the FF domain of HYPA/
FBP11, which is in equilibrium with <2% of unfolded
conformation. The PCS-CEST experiment reproduced these
results. In particular, the experiments showed that the PCSs of
the minor species were small, as expected for a less-ordered
conformation.
5.2. Biomolecules with Paramagnetic Tags in the Solid
State
The protein GB1 has been investigated extensively as a model
protein to test the effect of cysteine ligated paramagnetic tags by
MAS solid-state NMR.
642
Longitudinal R1PREs of 15N spins in
GB1 were measured in the presence of SPy-EDTA (Figure 7,
60)
248
loaded with Cu2+,
643
and also with Mn2+.
644
The PRE
data proved to be good indicators of distance and were measured
for 6 tagging sites and used as structural restraints for
computationally solving the protein structure,
645
while also
accounting for tag flexibility.
646
A less flexible short-armed
cyclen based tag TETAC (1-(2-(pyridin-2-yldisulfanyl)ethyl)-
1,4,7,10-tetraazacyclododecane) (Figure 9,112) was also
investigated with Cu2+ in GB1.
388
While the relatively short
T1e time of Cu2+ ions results in appreciable R1PREs by the SBM
mechanism, R2PREs are often small, which presents the basis for
increasing the sensitivity of NMR experiments by accelerating
the recovery of longitudinal magnetization between scans with
little increase in line width.
647
R1PREs of 1H spins are
challenging to measure in solids due to spin diffusion effects, but
R2PREs of 1H can offer long-range distance restraints.
648
R2
PREs by the SBM mechanism are largest for paramagnetic
centers with slowly relaxing electrons such as the nitroxide spin
label, which has also been demonstrated in GB1 by ligating
cysteine residues with MTSL.
649
Measurement of PCSs
generated by paramagnetic metal tags is more difficult in solids
than in solution, because different orientations of the metal
complex relative to the protein would lead to a range of different
PCSs. Nonetheless, PCSs have successfully been measured in
GB1 ligated with 4MMDPA tag (Figure 11,116) loaded with
Co2+ and lanthanoids and were used to help solve the protein
structure with Rosetta.
389
Because of the close proximity of molecules in the solid state,
intermolecular paramagnetic effects can be substantial. To
minimize these, in most investigations of paramagnetic GB1 in
the solid state the sample was diluted with diamagnetic protein.
Alternatively, intermolecular paramagnetic effects in a purely
paramagnetic sample can be accounted for by methods of NMR
crystallography that consider paramagnetic effects in a periodic
lattice.
650
Aside from solid proteins in the microcrystalline state,
paramagnetic effects have also been investigated by solid-state
NMR of membrane proteins. For example, the seven-helix
transmembrane protein sensory rhodopsin from Anabaena
tagged at two sites with MTSL nitroxide spin labels was
investigated in lipid bilayers.
651,652
Intermolecular PREs arising
from the nitroxide spin label revealed the oligomerization
interface of the monomer. Another membrane system, lactose
transport protein from Streptococcus thermophilus tagged with 3-
maleimido-proxyl (Figures 5 and 14),
211
was investigated in
native membranes.
653
Here, PREs were measured for a 13C-
labeled substrate to probe the proximity of an interhelix loop to
the protein active site. Finally, PREs have also been utilized to
solve the structure of a precipitated protein−RNA complex,
named L7Ae, by using IA-PROXYL spin label (Figures 5 and
17)
213
at various tagging sites of the protein and measuring
intermolecular PREs in isotope labeled RNA.
654
6. APPLICATIONS OF DIFFERENT TYPES OF
PARAMAGNETIC PROBES
Table 4 presents an overview of synthetic probes that have been
successfully applied to protein studies by NMR spectroscopy. It
provides an impression of the range of applications, where the
various probes have been found to be useful. The table also
includes some of the probes developed with the aim to obtain
narrow distance distributions in DEER experiments. This
section briefly summarizes the applications of the different
probes listed. Examples that were already mentioned in section 5
are not discussed in detail again here.
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AT

6.1. Applications of Nitroxide Probes
Commercially available MTSL (Figure 3) has been used
successfully in a wide range of protein NMR studies, making it
impossible to compile all usage in a comprehensive way.
Methods have been devised to speed up accurate 1H PRE
measurements
655
and been applied to demonstrate that MTSL
derivatized with a bulky group sufficiently limits the internal
motions of the tag to enable the PRE data analysis with the
assumption of a single nitroxide position relative to the
protein.
219
The potential of PREs generated by MTSL for
global fold determination of proteins was assessed by Battiste
and Wagner, who showed their value for determining the fold of
eukaryotic translation initiation factor 4E (eIF4E) in combina-
tion with HN−HNNOEs and chemical shift data.
130
Choosing
Bacillus amyloliquefaciens barnase as the model protein,
Gaponenko et al. also provided an early demonstration of the
value of MTSL-generated PREs in improving the accuracy and
ease of global folding determination.
656
The long-range nature
of PREs (generated by MTSL) was shown to be particularly
valuable for determining the structure of a loop-rich protein,
SAPK-interacting protein 1 (Sin1) in combination with limited
NOE data.
657
Examples for the use of MTSL in NMR studies of protein−
protein complexes have been mentioned in section 5.
65,91,658
In
a recent example, Olivieri et al. used a mixture of 15N- and
MTSL-labeled ubiquitin with 15N/13C-labeled ubiquitin to
distinguish intra- and intermolecular PREs in the transient
diubiquitin complex in a single sample.
659
MTSL has also been
used more generally in NMR studies of complexes between
proteins and smaller ligands. Gochin and co-workers employed
paramagnetic NMR in a second-site screening campaign
660
to
identify ligands binding in the hydrophobic pocket of HIV-1
gp41.
661
By labeling a short peptide, which was known to bind at
the edge of the pocket, with MTSL or cysteaminyl-EDTA
loaded with either Fe(II) or Co(II), the authors measured the
paramagnetic effects on the low-molecular weight ligand as it
was bound to the protein, thus detecting its binding site.
661
The
MTSL labeled peptide alone provided sufficient information as
the same group, in a subsequent study, used only MTSL labeled
peptide to determine the binding site of a different indole ligand
binding to HIV-1 gp41.
662
PREs measured with Rho guanine-
nucleotide dissociation inhibitor 2 (RhoGDI2) labeled with
MTSL provided Ruan and co-workers with key structural
information to characterize the binding mode of three hits
identified from a fragment library of 890 compounds.
663
The strong distance dependence of PREs generated by MTSL
has been successfully exploited in studies of protein dynamics,
including studies of the interdomain movements of cardiac
troponin C
664
and calmodulin.
665
Using a mixture of MTSL
labeled and isotope labeled protein, Yang et al. monitored the
slow exchange of monomeric units in the homodimer Dsy0195
from Desulfitobacterium hafniense Y51 by paramagnetic NMR
and DEER experiments.
666
Investigation of the open-to-closed
transition of maltose-binding protein with PREs generated by
the probe maleimide-TEMPO (Figure 5,15) revealed that the
apo state of the protein populates a major open form and a minor
closed species.
667
MTSL has also been utilized in NMR studies of proteins in the
solid state,
649,652,668,669
where structural restraints from PREs
can amount to a sizable fraction of the data available for structure
calculations. Especially in the case of membrane proteins, MTSL
has proven useful to assess their dynamics,
670
structure,
586,671
and function.
672
Using the MTSL tagged and cyanobacterial lectin CV−N-
containing fluorinated amino acids as an example, Matei and
Gronenborn demonstrated that the high resolution of 19F NMR
spectra allows facile measurement of the distances between 19F
spins and the paramagnetic center by 19F PREs, which proved to
be in a good agreement with the results from 1H PRE
measurements.
587
Following labeling of a Myc peptide with a
CF3compound, 19F PREs were also shown to provide a
convenient way of assessing proximity to MTSL labeled Max
peptide, both in the disordered state and the structurally defined
complex with DNA.
673
6.2. Applications of Aminopoly(Carboxylic Acid)-Based
Probes
Dvoretsky et al.
674
and Vadim et al.
250
showed that SPy-EDTA
(Figure 7,60) complexed with different paramagnetic metal ions
enables structural studies of proteins by paramagnetic NMR.
Because of the different enantiomeric forms of EDTA−metal
complexes, the tag is suited for PRE measurements but is less so
for PCS measurements, as demonstrated in a structure
refinement of the protein domain ArgN using the SPy-EDTA-
Cu(II) complex.
249
Nonetheless, SPy-EDTA loaded with
different lanthanoid ions proved successful in aligning a helical
transmembrane protein for RDC measurements.
675
As already
discussed in section 5.2,SPy-EDTA loaded with Mn(II)
644
and
Cu(II)
643
was also successfully applied to probe the structure of
GB1 by solid-state NMR. Using solid-state NMR, Wang et al.
identified glucuronoarabinoxylan, which is the major matrix
polysaccharide in maize cell walls, as the binding target of β-
expansins, by tagging the protein with Mn(II)-SPy-EDTA and
measuring 1H and 13C PREs in 13C enriched maize cell walls.
676
4MMPyMTA (Figure 7,73), 4PS-PyMTA (Figure 7,76),
and 4PS-BrPyMTA (Figure 7,77) are three other frequently
used probes employed in protein NMR research. 4PS-PyMTA
and 4PS-BrPyMTA were used for elucidating the 3D structure
of a protein in cells
610
and in pure protein solution (section
5.1.2).
266
Su and co-workers labeled SrtA with 4PS-BrPyMTA
to assign NMR signals of an unstable intermediate by using
PCSs and residue-selective isotope labeling.
677
4MMPyMTA
was used to detect the excited state of a protein−peptide
complex by the new PCS-CEST method (section 5.1.5),
263
and
this approach was recently extended to 19F NMR, referred to as
19F PCS-CEST, to gain PCS restraints for protein−ligand
interactions in the intermediate exchange regime.
678
In a similar
vein, 1H PCSs of a protein−ligand complex presenting a minor
species in solution were extracted from relaxation dispersion
experiments (1H PCS-RD experiments) to gain insight into the
binding mode of a ligand to a bromodomain labeled with a
4MMPyMTA−Tm(III) tag.
679
The approach elegantly circum-
vents excessive line broadening in the intermediate exchange
regime by detecting the PCSs of the minor species in the NMR
signals of the ligand added in large excess.
679
6.3. Applications of Cyclen-Based Probes
Section 5 gave some examples of applications of cyclen based
probes in protein studies. Further examples are summarized
below.
6.3.1. Double-Anchored Probes. The encounter complex
between Cc and bovine adrenodoxin (Adx) was characterized by
measuring PCSs and RDCs generated by the double-arm probe
Yb(III)-CLaNP-5 (Figure 9,79)onCc.
76
Loaded with different
lanthanoid ions, CLaNP-5 also delivered highly informative
PCSs and PREs to model the structure of the little soluble 65
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AU

kDa complex between membrane associated Adx and
adrenodoxin reductase (AdR).
680
Saio et al. used Yb(III)-CLaNP-5 to explore ligand-driven
conformation changes of the multidomain protein MurD, where
the PCSs identified a novel semiclosed conformation of the
protein.
617
Venkata et al. used PREs generated by Gd(III)-
CLaNP-5 and MTSL to provide compelling evidence for two
fundamentally different orientations of polyproline helices when
bound to the Src SH3 domain, which are populated to different
degrees.
681
The CLaNP-5 tag has also proven useful to investigate
conformational changes of proteins. In a maximum occurrence
(MO) analysis of the different relative domain orientations of
CaM, PCS, and PRE data generated by Ln(III)-CLaNP-5 tags
attached to either domain of CaM significantly improved the
definition of the conformational space populated by the
protein.
682
This tag was also used to study domain orientation
in matrix metalloproteinase-1.
616
More recently, Yb(III)-
CLaNP-5 was used to investigate the conformational states of
the single-domain GH11 glycosidase from Bacillus circulans
(BCX)), which proved to be in full agreement with crystal
structures
683
and only locally affected by site-directed muta-
genesis.
684
The double-anchored probe CLaNP-7 (Figure 9,80) proved
similarly useful for the determination of the structure and
encounter state of the complex between cytP450cam and
putidaredoxin (Pdx), as discussed in section 5.1.3.
629,631
Paramagnetic data recorded with the Yb(III)-CLaNP-7 tag
further demonstrated that the structure of the cytP450cam−Pdx
complex predominantly populates a closed conformation in
solution in contradiction to the open state reported by other
crystal structures.
685
CLaNP-7 also proved useful to generate
paramagnetic structure restraints of the heat-shock protein 90
(Hsp90) in the presence of proteins bound to the
chaperone.
686,687
Except for a recent study on domain motions in
diubiquitin,
614
the use of other double-arm probes, such as
T1/T2 (Figure 9,82)
688
and CLaNP-13 (Figure 9,85)
281
has
only been explored for DEER distance measurements between
two Gd(III) ions, with the aim to obtain narrow distance
distributions by restricting the flexibility of the tags. In either
case, the widths of the distance distributions obtained was not
narrower than those obtained with some of the singly linked
probes.
6.3.2. Applications of Single-Arm Cyclen Probes.
Among the single-arm cyclen based probes, DOTA-M8 (Figure
9,86) and C1/C2 (Figure 9,96) have been applied most
frequently for protein structure investigations. The probe C2
loaded with Dy(III) and Yb(III) attached at different sites was
used to assign the methyl resonances of a four-helix bundle
SNARE complex by measuring PCSs in 13C-HMQC spectra.
689
PCSs induced in the C2B domain of synaptotagmin-1 bound to
the SNARE complex provided clear evidence for a highly
dynamic binding mode between the two components.
690
The dengue virus serotype 2 NS2B-NS3 protease (NS2B-
NS3pro) is a recognized drug target but yields an overlapped
and difficult to assign NMR spectrum. The interaction of NS2B
with NS3pro was investigated by PCSs generated with probes
C1 and C2 loaded with Tm(III) and Tb(III). The PCSs
revealed that the enzyme predominantly assumes a closed
conformation that is stabilized by the presence of positively
charged active-site inhibitors.
291,618
The same probes were used
to investigate a NS2B-NS3pro construct without covalent
linkage, which confirmed the closed conformation in the
absence of inhibitors.
619
A similar situation was documented
for a linked construct of the related Zika virus NS2B-NS3
protease, where PCSs were induced in NS2B by Tb(III)-C2 and
Tm(III)-C2 attached to NS3pro, revealing the closed
conformation and stabilization by the presence of inhibitor.
691
PCSs generated by the C1 and C2 tags also provided the basis
for the development of algorithms to determine the 3D structure
of proteins, using the PCSs of backbone amides and backbone
chemical shifts as the sole experimental restraints.
94,604,606,612
Specifically, Pearce et al. demonstrated that two tagging sites can
suffice to structurally define large segments of a protein,
provided PCSs are available for multiple homo- and
heteronuclear spins of the polypeptide backbone.
94
Using the
combined information from PCSs and RDCs, the same study
concluded that the molecular dynamics ensemble representation
of ubiquitin (PDB ID 2KOX)
692
fitted the paramagnetic NMR
data better than any single 3D structure available of the protein.
Finally, the study showed that 1H PCSs from tags at two sites in
many cases suffices to determine the side-chain conformations
of amino acids with methyl groups. In general, however, a
complete 3D structure determination of a protein requires at
least three, if not four, different probe tagging sites.
The C1 tag loaded with Yb(III) and Tb(III) ions was also
used to assess the binding mode of 15N/13C-labeled dGMP to
the nucleotide binding R3H domain from human Sμbp-2, as
well as the structural changes in the R3H domain in response to
nucleotide binding.
693
As an alternative to isotope labeling, a
chemical tag, such as a tert-butyl group, also enables the selective
detection of 1H NMR signals of stably bound ligands and has
been used to characterize the binding mode of a ligand to
dengue virus NS2B-NS3pro by PCSs generated with C2 loaded
with Tm(III) and Tb(III) and detected in NOESY spectra.
635
The DOTA-M8 tag (Figure 9,86) has been used in a
particularly diverse range of applications, including the improve-
ment of NMR spectral resolution of intrinsically disordered
proteins (IDP),
694
generation of RDCs to elucidate the
structure of a transmembrane helix
695
and an α-helix from
myosin-VI that is remarkably stable in solution as a single
helix,
696
generation of RDCs in the ribosomal stalk protein bL12
to probe the orientational independence of N- and C-terminal
domains,
697
determining the fluorine coordinates of ligands
bound to hCA II by PCSs measured in 19F NMR spectra
recorded with four different tag attachment sites (including
PCSs generated with M7PyThiazole-DOTA),
698
and gener-
ation of PCSs in RNA.
699
Specifically, Göbl et al. demonstrated
that the relatively weak paramagnetism of Yb(III) in DOTA-M8
was sufficient to improve spectral resolution in two different
IDPs and enable additional resonance assignments,
694
whereas
Chiliveri et al. used Tm(III)-DOTA-M8 to generate sizable
molecular alignment of the HIV-1 gp41 transmembrane helix.
695
Similarly, Wang et al. used Tm(III)-DOTA-M8 to assess the
domain alignment of ribosome-bound bL12 monomers and
dimers by RDC measurements,
697
Zimmermann et al. used the
Tm(III)-DOTA-M8 tag to generate PCSs in 19F NMR spectra
of fluorine containing ligands bound to hCA II,
698
and
Strickland et al. showed that the RNA-binding protein U1A
tagged with Tm(III)-DOTA-M8 is capable of generating sizable
PCSs in an RNA hairpin equipped with a U1A binding loop,
which would otherwise be difficult to label with a paramagnetic
tag.
699
M7PyThiazole-DOTA (Figure 9,92) is a newer probe than
DOTA-M8, which offers a more rigid tether between lanthanide
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AV

ion and protein backbone. Loaded with Tm(III) and attached to
each monomer of the homotrimeric protein Skp, a single set of
PCSs was generated by M7PyThiazole-DOTA, which could be
fitted by Δχtensors positioned at three sites related by C3
symmetry.
700
Using DOTA-M8 and M7PyThiazole-DOTA as
paramagnetic tags, Cucuzza et al. developed a new iterative
procedure for structure refinement of repeat proteins based on
backbone amide PCSs. The authors successfully determined the
fold of the protein YM4A in solution.
701
The related probes
M7Py-DOTA (section 5.1.2)
286
and M8-CAM-I,
285
which
likewise generate stable thioether linkages with cysteine, have
been deployed for in-cell measurements of PCSs to obtain
structural restraints with Tm(III) and Dy(III) ions, respectively,
in GB1 and ubiquitin.
Lanthanoid tags can be deployed in DEER experiments when
loaded with Gd(III), while generating PCSs in NMR experi-
ments when loaded with other paramagnetic lanthanoid ions.
Both approaches were combined by Abdelkader et al. in a
structural analysis of the E. coli aspartate/glutamate binding
protein with the C3 tag ligated to AzF.
702
Multiple sites tagged
pairwise with Gd(III)-C3 delivered accurate positions of the
metal ion relative to the crystal structure of the protein, which
subsequently allowed fitting Δχtensors using PCSs measured
with Tm(III)-C3 and Tb(III)-C3 in 5-parameter fits that relied
on the metal coordinates derived from the DEER measure-
ments. The approach is of interest for proteins of limited
solubility, where resonance assignment by 3D NMR experi-
ments is difficult but 2D NMR spectra are accessible of samples
prepared with selective isotope labeling, which can be assigned
by high-throughput site-directed mutagenesis.
Gd(III) complexes of the above-mentioned probes and other
single arm probes have been explored for distance measure-
ments by DEER experiments in multiple EPR studies.
298,703−709
Specifically the probes DO3MA-3BrPy (Figure 9,107) and C3
(Figure 9,98) were deployed for distance measurements in
proteins embedded in cells.
708,709
6.4. Applications of Small Chemical Probes
Compared with the aforementioned probes, small chemical
based probes have been less frequently used in paramagnetic
NMR of proteins, which can be attributed to the inconvenience
of having to titrate with paramagnetic metal ions after the
tagging reaction and the risk of populating alternative binding
sites with stray metal ions, which do not bind at the intended site
either due to weak binding affinities to the probe or an excess of
metal ion. Among the small chemical Gd(III) probes,
4MMDPA (Figure 11,116) and the IDA-SH (Figure 11,
126) tag have been used for DEER distance measurements in
proteins.
688,710
6.5. Applications of Cosolute Paramagnetic Probes
Cosolute paramagnetic probes are popular tools for detecting
solvent accessible protein surfaces. Petros et al. explored the use
of TEMPOL and [Gd(III)(DTPA)]2−for the identification of
solvent exposure of ubiquitin and hen egg white lysozyme
(HEWL) by double-quantum-filtered proton correlation spectra
(DQF-COSY), concluding that the two probes preferentially
interacted with hydrophobic amino acid side chains and
carboxylate groups, respectively.
345
Fesik et al. subsequently
showed that longitudinal 1H PREs generated by TEMPOL
identified the solvent-exposed residues of 13C-labeled cyclo-
sporin bound to cyclophilin.
346
The same group confirmed this
approach using a 13C-labeled FK-506 analog bound to FKBP.
711
Molinari et al. concluded that TEMPOL makes no specific
interactions with bovine pancreatic trypsin inhibitor (BPTI),
allowing interpretation of signal attenuations in homo- and
heteronuclear NMR spectra in terms of solvent exposure.
712
This was followed by a string of paramagnetic perturbation
studies of a number of proteins, including Tendamistat,
713
MNEI,
346
HEWL,
347,355
and Sso7d (from Sulfolobus solfatar-
icus),
714
using TEMPOL, as well as soluble Gd(III) complexes
as the paramagnetic agents. Bernini et al. investigated protein
aggregation by monitoring the attenuation of 1H NMR signals of
BPTI in the presence of TEMPOL and Gd(III)DTPA-BMA as
a function of protein concentration.
715
TEMPOL-induced
paramagnetic perturbation was shown to be broadly compatible
with results obtained on the solvent exposure of aromatic amino
acid side chains by photochemically induced dynamic nuclear
polarization (photo-CIDNP) experiments.
347
The introduction
of Gd(III)DTPA-BMA provided an alternative uncharged
cosolute probe for paramagnetic perturbation studies, which is
less hydrophobic than TEMPOL and was shown to deliver
better correlations between calculated and experimental solvent
PREs
350
but in the case of α-bungarotoxin was reported to still
show uneven attenuations of [13C,1H]-HSQC cross-peaks of
CαH groups across the protein,
716
which were reproduced by the
much larger probe Gd2L7.
717
To identify the interaction surface
of a protein−protein complex by comparing cross-peak
attenuations of the complex versus those of the individual
proteins, a charged probe like [Gd(III)(EDTA)]1−was shown
to be sufficient.
357
Almeida et al. further demonstrated that also
the highly charged probes [Gd(III)(DOTP)]5−and [Gd(III)-
DOTAM]3+ allow probing for protein−protein interactions.
718
It has been noted, however, that Gd(III) complexes with free
coordination sites, such as in Gd(III)(DO3A), can favor
transient associations with carboxylate groups on the protein
surface.
366
Different algorithms have been developed to exploit PREs
from paramagnetic cosolutes for determining the 3D structures
of proteins
352
and protein complexes,
719
as well as gaining
information on protein dynamics.
353
Madl et al. selected
Gd(III)DTPA-BMA to aid protein structure determination,
using a “second-sphere interaction model”to translate PREs into
distance restraints between the NMR-active nuclei and the
protein surface.
352
This became the basis of a strategy to
integrate solvent PRE data generated by Gd(III)DTPA-BMA
into the program HADDOCK
720
to elucidate the structure of a
150 kDa ternary protein complex. Gd(III)DTPA-BMA also
provided useful PRE restraints for the structure determination of
the homodimeric protein Sam68,
721
and the probe clearly
demonstrated how farnesylation alters the solvent accessibility
of the C-terminal polypeptide segment of peroxisomal bio-
genesis factor 19 (PEX19).
722
By measuring transverse PREs of
amide protons with Gd(III)DTPA-BMA as a function of
protein concentration and taking into account local molecular
crowding as an impediment to translational diffusion, Jens Led
and co-workers identified a method to distinguish specific from
nonspecific contacts in weakly self-associating human growth
hormone.
723
To eliminate exchange effects arising from rapid
relaxation of Gd(III)-coordinated hydration water, the group of
Chun Tang developed Gd(III)TTHA-TMA as a cosolute probe
without free coordination sites on the Gd(III) ion, which
allowed determining the populations of open and closed
conformations from solvent PREs by comparison with
theoretical expectations based on crystal structures and
molecular dynamics simulations.
353
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AW

As discussed in section 3.3.5, the [Ln(III)(DPA)3]3−probe
can generate measurable PCSs and its Gd(III) complex can
produce PREs. Ma et al. investigated the binding of [Ln(III)-
(DPA)3]3−probes (Ln = Eu, Gd, Tb, Tm, or Yb) to multiple
sites in HEWL, which had been identified in a crystal structure of
the protein with [Eu(III)(DPA)3]3−.
724
While the para-
magnetic effects observed could not be fully accounted for by
the crystal structure, an MD simulation reproduced the highest
affinity binding site and correlated small PCS magnitudes with
increased local protein dynamics. More recently, Swarbrick et al.
used PCSs generated by [Tm(III)(DPA)3]3−to improve the
spectral resolution of NOESY spectra of lipopeptide polymyxin
B (PMXB) and the peptide MSI-594 bound to micelles.
725
The
PCSs originated from transient association of the probe with the
peptides, but were not used as structural restraints. Instead, the
concentration of the probe was tuned to improve the spectral
resolution while limiting PREs, and the structures were
determined from trNOEs.
Finally, it has been pointed out that, at a fundamental NMR-
technical level, paramagnetic solvent probes can be used to
simplify NMR spectral interpretation. For example, Schwar-
zinger and co-workers showed that difference spectra calculated
for HSQC or NOESY experiments recorded in the presence and
absence of Gd(III)DTPA-BMA can be used to focus on signals
with little accessibility to the probe.
726
Similarly, Sattler and
Fesik proposed the use of high concentrations of [Dy(III)-
(DTPA)]2−to obtain substantial chemical shift changes in
NMR spectra.
358
7. CONCLUSIONS AND PROSPECTS
The invention and further development of paramagnetic and, in
particular, site-specific probes has opened a wide range of
protein structure investigation methods both by NMR spec-
troscopy and EPR spectroscopy. Although nitroxide spin labels
have long been used with great success for PRE measurements
and in EPR studies, many more structure restraints beyond
distances become accessible with paramagnetic metal ions
associated with anisotropic molecular magnetic susceptibility
tensors. Lanthanoid ions stand out for their intrinsically large Δχ
tensors and synthetic chemists have worked hard to optimize
lanthanoid binding probes. A number of broad conclusions can
be drawn from comparing the performance of these probes. (i)
Site-specific attachment is difficult to attain without one or more
covalent bonds. (ii) Rigid attachment of a lanthanoid ion is
important for obtaining useful Δχtensors and more easily
achieved by double-arm attachment. In the case of proteins,
immobilization can be achieved by additional coordination by
carboxylate groups of amino acid side chains. (iii) Probes
synthesized with the lanthanoid ion in place are more user-
friendly and less prone to artifacts than probes that need to be
titrated with metal ions following ligation with the target
molecule. Therefore, cyclen based probes continue to be
attractive despite the multistep protocols of their synthesis.
Significant challenges remain. Most covalently binding probes
target cysteine residues and thus are incompatible with proteins
containing natural cysteine residues that are accessible and
functionally important. To avoid the dependence on cysteine
residues, schemes for site-specific incorporation of noncanonical
amino acid capable of binding lanthanoid probes have been
developed. A generally satisfying solution has not yet been
found, however, because either the resulting linker to the target
protein is quite long (as in the case of, for example, probes
attached by click chemistry), the unnatural amino acid disrupts
the protein structure (as in the case of, for example,
phosphoserine), or the amino acid causes protein precipitation
upon metal binding (as in the case of the bipyridyl and 8-
hydroxy-quinoline amino acids). An emerging solution may be
offered by the site-specific incorporation of photocaged
selenocysteine, which, following decaging by UV irradiation,
delivers a site that is much more reactive toward alkylating
probes than cysteine thiol groups.
727,728
The potential of
harnessing this strategy for attaching double-armed probes is
clear but has not yet been explored. Also the incorporation of an
unnatural amino acid containing a stable nitroxide radical has
not yet been achieved in a way that would dethrone the MTSL
tag as the most popular spin label, partly because nitroxides are
sensitive to reduction in live cells and partly because no
aminoacyl-tRNA synthetase is available that is specific for a
nitroxide amino acid featuring a short and rigid linker between
nitroxide group and protein backbone.
Moving from lanthanoid ions to transition metal ions reduces
the size of complexation cages and enables good binding
affinities with nitrogen ligands but abandons the large and varied
range of paramagnetic effects afforded by different lanthanoid
ions. Attachment modes that leave vacant coordination sites on
the metal ion risk leading to metal mediated protein
dimerization. While this has not been observed routinely, it is
a distinct possibility for many of the very small probes and also
when a transition metal ion binding site is engineered by a pair of
histidine residues.
With the advent of completely rigid probes, exciting prospects
are opened for high-accuracy protein structure analysis,
including determining the 3D structures of minor, little
populated protein states by PCSs. With chemically stable
ligation chemistry, such studies will foreseeably be possible also
in living cells. Therefore, finding new rigid chelators remains an
important goal for the future.
Finally, paramagnetic metal probes are becoming increasingly
popular in EPR spectroscopy for measuring metal−metal
distances in the nanometer range, where the metal ions can be
Gd(III), Mn(II), or Cu(II) ions.
121,162,163,729
Both NMR and
EPR are based on spin interactions, and it is only logical that
paramagnetic systems bring these two fields closer together. The
combination of these spectroscopies provides a more complete
picture of the world of biological macromolecules.
AUTHOR INFORMATION
Corresponding Author
Marcellus Ubbink −Leiden Institute of Chemistry, Leiden
University, Leiden 2333 CC, The Netherlands; orcid.org/
0000-0002-2615-6914; Email: m.ubbink@
chem.leidenuniv.nl
Authors
Qing Miao −Leiden Institute of Chemistry, Leiden University,
Leiden 2333 CC, The Netherlands; School of Chemistry
&Chemical Engineering, Shaanxi University of Science &
Technology, Xi’an 710021, China; orcid.org/0000-0003-
0587-1916
Christoph Nitsche −Research School of Chemistry, The
Australian National University, Canberra, Australian Capital
Territory 2601, Australia; orcid.org/0000-0002-3704-
2699
Henry Orton −Research School of Chemistry, The Australian
National University, Canberra, Australian Capital Territory
2601, Australia; ARC Centre of Excellence for Innovations in
Chemical Reviews pubs.acs.org/CR Review
https://doi.org/10.1021/acs.chemrev.1c00708
Chem. Rev. XXXX, XXX, XXX−XXX
AX

Peptide &Protein Science, Research School of Chemistry,
Australian National University, Canberra, Australian Capital
Territory 2601, Australia
Mark Overhand −Leiden Institute of Chemistry, Leiden
University, Leiden 2333 CC, The Netherlands
Gottfried Otting −Research School of Chemistry, The
Australian National University, Canberra, Australian Capital
Territory 2601, Australia; ARC Centre of Excellence for
Innovations in Peptide &Protein Science, Research School of
Chemistry, Australian National University, Canberra,
Australian Capital Territory 2601, Australia; orcid.org/
0000-0002-0563-0146
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.chemrev.1c00708
Notes
The authors declare no competing financial interest.
Biographies
Qing Miao completed her Ph.D. under the supervision of Prof.
Marcellus Ubbink and Dr. Mark Overhand in 2019 at Leiden
University. During her Ph.D. studies, she focused on paramagnetic
NMR probe design, synthesis, and applications. Currently, she works as
a junior research fellow at Shaanxi University of Science & Technology
and is interested in natural abundance isotope analysis research.
Christoph Nitsche studied chemistry and business administration and
received his Ph.D. in medicinal chemistry from Heidelberg University
in 2014. He worked as a Feodor Lynen Fellow at the Australian
National University and as a Rising Star Fellow at the Free University of
Berlin. In 2020, he was appointed at the Australian National University,
where he is currently Senior Lecturer and ARC DECRA Fellow in the
Research School of Chemistry. His research program in medicinal
chemistry and chemical biology focuses on drug discovery against
infectious diseases, biocompatible chemistry, and peptide and protein
modification.
Henry Orton completed his Ph.D. in 2020 at the Australian National
University with a focus on experiment and software development for
paramagnetic relaxation in biomolecular NMR spectroscopy. He is
currently a postdoctoral fellow with Professor Gottfried Otting with
interests in ultrafast magic angle spinning of biological solids.
Mark Overhand obtained a M. Sc. (Dec. 1989) from Leiden University
(oligosaccharide synthesis, supervisors G.J. Boons, G. van der Marel,
and J.H. van Boom) and a Ph.D. (Jan. 1995) from the University of
Virginia, US (natural product synthesis, advisor S.M. Hecht). He did a
postdoc. with a Swiss stipend at the ETH in Switzerland in the group of
D.S. Seebach (design and synthesis of novel beta-hexapeptide helix)
and returned to Leiden where he remains (1996−present): postdoc,
assistant prof. (UD), associate prof. (UHD). Stanford: sabbatical
(summer 2004). Awards: Golden spatula 1986 (B. sc. research), best
teacher faculty of science 2006, Leiden University.
Gottfried Otting studied chemistry at the universities of Heidelberg and
Freiburg (Germany) and received his Ph.D. in protein NMR
spectroscopy from the ETH-Zürich (Switzerland) in 1987. He
continued at the ETH-Zürich as a research assistant until 1992, when
he was appointed professor of Molecular Biophysics at the Karolinska
Institute in Stockholm (Sweden). In 2002, he was appointed professor
at the Research School of Chemistry of the Australian National
University in Canberra (Australia) on a Federation Fellowship by the
Australian Research Council (ARC). He currently is an ARC Laureate
Fellow at the same place. His research program focuses on method
developments for protein NMR spectroscopy, including techniques for
the production of protein samples with labels for structure analysis and
drug discovery.
Marcellus Ubbink obtained a Ph.D. in Chemistry from Leiden
University in 1994, working on paramagnetic properties of metal-
loproteins. During his postdoc research at the University of Cambridge
(UK), he developed a method to determine the structure of two
proteins using paramagnetic NMR, based in intermolecular para-
magnetic effects caused by a heme group. He returned to Leiden
University in 1997 as an assistant professor and was appointed to
associate professor (2004) and full professor (2010). Since 2019, he is
also Programme Director of the bachelor Life Science and Technology.
His current research interests comprise protein−protein interactions,
enzymology, protein evolution, and paramagnetic protein NMR
spectroscopy.
ACKNOWLEDGMENTS
Q.M. acknowledges support from the Chinese Scholarship
Council (CSC grant to Q.M., No. 201506870013). C.N.
acknowledges funding from the Australian Research Council
(DE190100015, DP200100348). Financial support by the
Australian Research Council for a Laureate Fellowship to G.O.
(FL170100019) and through a Centre of Excellence
(CE200100012) is also gratefully acknowledged.
ABBREVIATIONS
2AP 2-aminopyridine
AdR adrenodoxin reductase
ArgN Escherichia coli arginine repressor
BCX single-domain GH11 glycosidase from Bacillus
circulans
BPTI bovine pancreatic trypsin inhibitor
BpyAla bipyridyl alanine
BuLi butyllithium
CAP catabolite gene activator protein
Cc yeast iso-1-cytochrome c
CcP yeast cytochrome cperoxidase
CCR cross-correlated relaxation
CDI N,N′-carbonyldiimidazole
CEST chemical exchange saturation transfer
CLaNP caged lanthanoid NMR probe
COSY correlation spectroscopy
CPMG Carr−Purcell−Meiboom−Gill
CSA chemical shift anisotropy
CTAB cetyltrimethylammonium bromide
CuAAC copper-catalyzed azide−alkyne cycloaddition
Cyclen 1,4,7,10-tetraazacyclododecane
Cys-Ph-TAHA cysteinyl-phenyl-triaminohexaacetate
cytP450cam cytochrome P450cam
DCM dichloromethane
DD dipole−dipole
DEER double-electron−electron resonance
DIPEA N,N-diisopropylethylamine
DMF N,N-dimethylformamide
DMSO dimethyl sulfoxide
DNA desoxyribonucleic acid
DNP dynamic nuclear polarization
DOTA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra-
acetic acid
dPA 2,2′-dipicolylamine
DPA dipicolinic acid
DPAP 2,2-dimethoxy-2-phenylaceto-phenone
DQF-COSY double-quantum-filtered COSY
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AY

DSA dipolar shielding anisotropy
DTNB 5,5′-dithio-bis(2-nitrobenzoic acid)
DTPA diethylenetriaminepentaacetic acid
DTTA diethylene-triamine-tetraacetate
EDTA ethylenediaminetetraacetic acid
EdU 5-ethynyl-2′-deoxyuridine
eIF4E eukaryotic translation initiation factor 4E
EPR electron paramagnetic resonance
FAK focal adhesion kinase
FRET Förster resonance energy transfer
GB1 B1 immunoglobulin binding domain of protein
G
GPS global positioning system
HATU 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetrame-
thyluronium hexafluorophosphate
hCA II human carbonic anhydrase II
HEWL hen egg white lysozyme
HPPK Staphylococcus aureus 6-hydroxymethyl-7,8-di-
hydropterin pyrophosphokinase
HQA 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic
acid
Hsp90 heat-shock protein 90
HSQC heteronuclear single-quantum correlation
IDA iminodiacetic acid
IDP intrinsically disordered protein
IL1βinterleukin-1β
LBP lanthanoid-binding peptide
LDA lithium diisopropylamide
Ln(III) trivalent lanthanoid ion
Ln lanthanoid
m-CPBA meta-chloroperbenzoic acid
MAS magic angle spinning
MO maximum occurrence
MRI magnetic resonance imaging
MST methanethiosulfonate
MTSL (1-oxy-,2,2,5,5-tetramethyl-D-pyrroline-3-
methyl)-methanethiosulfonate
ncAA noncanonical amino acid
NMR nuclear magnetic resonance
NOE nuclear Overhauser effect
NOESY nuclear Overhauser effect spectroscopy
NS2B-NS3pro dengue virus serotype 2 NS2B-NS3 protease
NTA nitrilotriacetic acid
NTP nucleotide triphosphate
p75 ICD p75 neurotrophin receptor
PCS pseudocontact shift
PDB protein data bank
Pdx putidaredoxin
PEX19 C-terminal polypeptide segment of peroxiso-
mal biogenesis factor 19
photo-CIDNP photochemically induced dynamic nuclear
polarization
PPI protein−protein interactions
PRE paramagnetic relaxation enhancement
PyMTA 2,2′,2″,2‴-((pyridine-2,6-diylbis(methylene))-
bis(azanetriyl))tetraacetic acid
RACS residual anisotropic chemical shift
RADS residual anisotropic dipolar shift
RD relaxation dispersion
RDC residual dipolar coupling
RhoGDI2 Rho guanine-nucleotide dissociation inhibitor
2
RMSD root-mean-square deviation
RNA ribonucleic acid
SAP square antiprism
SBM Solomon−Bloembergen−Morgan
SH3 Src homology 3
SrtA Staphylococcus aureus sortase A
PMXB lipopeptide polymyxin B
T4Lys T4 lysozyme
TAHA triaminohexaacetate
TCEP tris(2-carboxyethyl)phosphine
TETAC 1-(2-(pyridin-2-yldisulfanyl)ethyl)-1,4,7,10-
tetraazacyclododecane
THF tetrahydrofuran
TMR tetramethylrosamine
TMS trimethylsilyl
TMSCHN2(diazomethyl)trimethylsilane
tPCS transferred PCS
tPRE transferred PRE
TPS 2,4,6-triisopropylbenzenesulfonyl
TraNP transition metal ion NMR probe
TROSY transverse relaxation optimized spectroscopy
TSAP twisted square antiprism
Symbols
χmagnetic susceptibility tensor
χxmagnetic susceptibility value along the tensor framework
xaxis
χymagnetic susceptibility value along the tensor framework
yaxis
χzmagnetic susceptibility value along the tensor framework
zaxis
Δχanisotropic magnetic susceptibility tensor
Δχax axial component of the Δχtensor
Δχrh rhombic component of the Δχtensor
μBBohr magneton
kBBoltzmann constant
Tabsolute temperature
μ0induction constant
geelectronic gfactor
Stotal electron spin quantum number
gJelectronic gfactor of lanthanoid ions
Jtotal angular momentum quantum number
σdipolar shift tensor
rvector connecting paramagnetic center and nuclear spin
r|r|

33×3 identity matrix
⊗Kronecker product
Aalignment tensor
B0magnetic field strength
γIgyromagnetic ratio of the nuclear spin I
γKgyromagnetic ratio of the nuclear spin K
rIK internuclear distance between Iand K
hPlanck’s constant
R1
SBM paramagnetic enhancement of the longitudinal relaxation
rate due to SBM mechanism
R2
SBM paramagnetic enhancement of the transverse relaxation
rate due to SBM mechanism
ωInuclear Larmor frequency
ωSelectron Larmor frequency
τccorrelation time
τrrotational correlation time
τselectronic lifetime
R1
Curie paramagnetic enhancement of the longitudinal relaxation
rate due to Curie mechanism
Chemical Reviews pubs.acs.org/CR Review
https://doi.org/10.1021/acs.chemrev.1c00708
Chem. Rev. XXXX, XXX, XXX−XXX
AZ

R2
Curie paramagnetic enhancement of the transverse relaxation
rate due to Curie mechanism
ΩLarmor frequency of the nuclear spin
σN1H spin shift tensor originated by the dipolar field of the
15N spin
σ↑positive 1H spin shift tensor originated by the dipolar
field of the 15N spin
σ↓negative 1H spin shift tensor originated by the dipolar
field of the 15N spin
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