No full-text available
To read the full-text of this research,
you can request a copy directly from the author.
NMR of proteins and Nucleic Acids
... The NMR structure determination of proteins is most typically based on inter-atomic proton-proton distances estimated from measured nuclear Overhauser effect (NOE). 13 As supplementary structural information, chemical shifts of backbone nuclei and occasionally scalar couplings or residual dipolar couplings (RDCs) 14 ...
... The CPMG experiments measured with σ 1:1 samples including stereospecifically labeled methyl groups provided us a complete set of 13 C methyl chemical shifts of the minor state. The most significant changes were identified for 13 C γ -proS of V15, 13 C δ -proS of L19, both methyls in I34, and 13 C δ -proR of L55. All these methyls are located in a proximity of aromatic rings in the major state ( 13 C γ -proS of V15 is close to F41, other mentioned methyls are in the proximity of Y51). ...
... where n was the number of available chemical shift disturbance for backbone nuclei in each residue and the parameters α i were 0.32, 0.19, 0.12, and 1.00 for 13 CO, 13 ...
σ $$ \sigma $$ factors are essential parts of bacterial RNA polymerase (RNAP) as they allow to recognize promotor sequences and initiate transcription. Domain 1.1 of vegetative σ $$ \sigma $$ factors occupies the primary channel of RNAP and also prevents binding of the σ $$ \sigma $$ factor to promoter DNA alone. Here, we show that domain 1.1 of Bacillus subtilis σ A $$ {\sigma}^A $$ exists in more structurally distinct variants in dynamic equilibrium. The major conformation at room temperature is represented by a previously reported well-folded structure solved by nuclear magnetic resonance (NMR), but 4% of the protein molecules are present in a less thermodynamically favorable state. We show that this population increases with temperature and we predict its significant elevation at higher but still biologically relevant temperatures. We characterized the minor state of the domain 1.1 using specialized methods of NMR. We found that, in contrast to the major state, the detected minor state is partially unfolded. Its propensity to form secondary structure elements is especially decreased for the first and third α $$ \alpha $$ helices, while the second α $$ \alpha $$ helix and β $$ \beta $$ strand close to the C-terminus are more stable. We also analyzed thermal unfolding of the domain 1.1 and performed functional experiments with full length σ A $$ {\sigma}^A $$ and its shortened version lacking domain 1.1 ( σ A _ Δ 1.1 $$ {\sigma}^{A\_\Delta 1.1} $$ ). The results revealed that while full length σ A $$ {\sigma}^{\mathrm{A}} $$ increases transcription activity of RNAP with increasing temperature, transcription with σ A _ Δ 1.1 $$ {\sigma}^{A\_\Delta 1.1} $$ remains constant. In summary, this study reveals conformational dynamics of domain 1.1 and provides a basis for studies of its interaction with RNAP and effects on transcription regulation.
... While nuclear magnetic resonance (NMR) is commonly appreciated for deciphering the structure of large molecules such as proteins [1,2], its potential to probe the mesoscopic structure of disordered media and biological tissues has yet to be fully realized. In this context, the mesoscopic structure is a few orders of magnitude coarser than molecules, but far below the macroscopic sample dimensions (Fig. 1). ...
... One of the main challenges for the mesoscopic NMR and MRI [16,24] is to develop a theory that enables extracting the mesoscopic features of materials or tissues from the measured signal, which would serve a role similar to that of the theory behind quantifying protein structure [1]. However, instead of the J-couplings of the quantum Hamiltonians for the interacting spins in the ''chemical" NMR [3,25], the mesoscopic NMR requires describing the effect of spatially disordered environments of composite materials and living tissues. ...
... This discussion shows that our main result expressed by Eqs. (1) and (2) is universal: The functional form of the relaxation timedependence reflects the scaling of the structural power spectrum for small k, which is independent of individual properties of the magnetized objects. This is illustrated in Appendix B, Fig. B.7, that shows results of MC simulations for the same media as in Fig. 2, but with the magnetized objects made impermeable for diffusing spins, as in our NMR measurement described in Section 2.6. ...
Nuclear magnetic resonance (NMR) has been instrumental in deciphering the structure of proteins. Here we show that transverse NMR relaxation, through its time-dependent relaxation rate, is distinctly sensitive to the structure of complex materials or biological tissues at the mesoscopic scale, from micrometers to tens of micrometers. Based on the ideas of universality, we show analytically and numerically that the time-dependent transverse relaxation rate approaches its long-time limit in a power-law fashion, with the dynamical exponent reflecting the universality class of mesoscopic magnetic structure. The spectral line shape acquires the corresponding non-analytic power law singularity at zero frequency. We experimentally detect the change in the dynamical exponent as a result of the transition into maximally random jammed state characterized by hyperuniform correlations. The relation between relaxational dynamics and magnetic structure opens the way for noninvasive characterization of porous media, complex materials and biological tissues.
... The spectra were assigned according to Wü thrich and used to create ensembles of low energy conformations of the peptide ( Figures 4B-4D). 36 In the case of the free peptide, the histidines pointed to opposite directions ( Figure 4B, root mean square deviation [RMSD] values: backbone 0.86 Å , heavy atoms 2.36 Å , and heavy atoms of His residues 1.39 Å ). After adding 1 molar equiv of Zn 2+ ions, the histidines moved closer in order to coordinate the metal ( Figures 4C and S10B). ...
... 60 Resonance assignment followed the sequential assignment methodology developed by Wü thrich. 36 The three-dimensional structures of the peptides were calculated using XPLOR-NIH (version 3.2) 61,62 by hybrid distance geometry-dynamical simulated annealing. Peak intensities were manually assigned as strong (2.5 Å ), medium (3.5 Å ), weak (4.5 Å ), and very weak (5.5 Å ) with a G0.5 Å error. ...
... 2D 1 H-NMR correlated spectroscopy (COSY), total correlation spectroscopy (TOCSY), and rotating frame Overhauser enhancement spectroscopy (ROESY) measurements were performed to determine the structure of the peptide and of the complex under aqueous and emulsion conditions. The spectra were assigned according to Wüthrich and used to create ensembles of low energy conformations of the peptide (Fig. 4b-e) 31 . In the case of the free peptide, the histidines pointed to opposite directions (Fig. 4b, RMSD values: Backbone 0.86 Å, heavy atoms 2.36 Å, heavy atoms of His residues 1.39 Å). ...
... Spectra were processed and analyzed with TopSpin (Bruker Analytische Messtechnik GmbH) and NMRFAM SPARKY software 50 . Resonance assignment followed the sequential assignment methodology developed by Wüthrich 31 . ...
Emulsions are commonly used for drug delivery, yet they are usually limited to exclusively delivering either lipophilic compounds or hydrophilic compounds. This separation negates possible synergetic therapeutic roles between such compounds. Here, we introduce a novel design for a short peptide that can stabilize emulsions. Upon binding certain metal ions, the peptide acts as a molecular switch, changes conformation, and becomes amphiphilic. Spectroscopic methods, NMR, and molecular dynamics provide information on the mechanism of this complexation-triggered amphiphilicity. The stability of these unique emulsions is based on histidine-metal bonds, which break at low pH values, selectively releasing their contents at the extracellular pH of tumors. Paclitaxel-encapsulated emulsion demonstrated strong activity against HeLa cells with an IC 50 of 70 nM, possibly enhanced by the simultaneous release of Zn ²⁺ ions. Importantly, the emulsion was easily functionalized with various hexahistidine-tagged motifs that can supply the emulsions with many functions beyond drug delivery.
... Spatial structure calculations were performed in the CYANA software package version 3.98.13 using the simulated annealing/molecular dynamics protocol [27]. Torsion angles restraints, stereospecific assignment and interproton distance restraints were obtained based on the J-couplings and 1 H-1 H NOE (nuclear Overhauser effect) connectivities. ...
Advancements in medicine and pharmacology have led to the development of systems that deliver biologically active molecules inside cells, increasing drug concentrations at target sites. This improves effectiveness and duration of action and reduces side effects on healthy tissues. Cell-penetrating peptides (CPPs) show promise in this area. While traditional medicinal chemistry methods have been used to develop CPPs, machine learning techniques can speed up and reduce costs in the search for new peptides. A predictive algorithm based on machine learning models was created to identify novel CPP sequences using molecular descriptors using a combination of algorithms like k-nearest neighbors, gradient boosting, and random forest. Some potential CPPs were found and tested for cytotoxicity and penetrating ability. A new low-toxicity CPP was discovered from the Rhopilema esculentum venom proteome through this study.
... The resonance assignments for the 1 H nuclei were obtained by analyzing TOCSY and NOESY spectra, as reported by Wuthrich. 66 The summary of structural statistics and quality analysis for the ensemble structures and the lowenergy structure can be found in Table 2. The 1 H− 1 H NOESY spectra analysis revealed 328 distance restraints for AMP-Cry10Aa and 291 restraints for AMPCry10Aa_5. ...
... The data were subsequently processed using Topspin 4.0.3 (Bruker, Billerica, MA, USA), with a solvent signal reference of 4.77 ppm at 298 K. Sequential assignment strategies [46] were used to assign and analyze the data in the program CARA (Computer Assisted Resonance Assignment) [47]. Additional TOCSY data at varying temperatures (280 K, 293 K, 298 K, 303 K) were recorded to monitor the temperature dependence of the amide protons. ...
Venom peptides have evolved to target a wide range of membrane proteins through diverse mechanisms of action and structures, providing promising therapeutic leads for diseases, including pain, epilepsy, and cancer, as well as unique probes of ion channel structure-function. In this work, a high-throughput FLIPR window current screening assay on T-type CaV3.2 guided the isolation of a novel peptide named ω-Buthitoxin-Hf1a from scorpion Hottentotta franzwerneri crude venom. At only 10 amino acid residues with one disulfide bond, it is not only the smallest venom peptide known to target T-type CaVs but also the smallest structured scorpion venom peptide yet discovered. Synthetic Hf1a peptides were prepared with C-terminal amidation (Hf1a-NH2) or a free C-terminus (Hf1a-OH). Electrophysiological characterization revealed Hf1a-NH2 to be a concentration-dependent partial inhibitor of CaV3.2 (IC50 = 1.18 μM) and CaV3.3 (IC50 = 0.49 μM) depolarized currents but was ineffective at CaV3.1. Hf1a-OH did not show activity against any of the three T-type subtypes. Additionally, neither form showed activity against N-type CaV2.2 or L-type calcium channels. The three-dimensional structure of Hf1a-NH2 was determined using NMR spectroscopy and used in docking studies to predict its binding site at CaV3.2 and CaV3.3. As both CaV3.2 and CaV3.3 have been implicated in peripheral pain signaling, the analgesic potential of Hf1a-NH2 was explored in vivo in a mouse model of incision-induced acute post-surgical pain. Consistent with this role, Hf1a-NH2 produced antiallodynia in both mechanical and thermal pain.
... The main contributions of this article are as follows: (1) In this paper, a cryo-EM clustering model based on contrast learning is proposed, which is used to complete the feature extraction task of unlabeled cryo-EM images, calculate similar features to generate pseudo-label data, and then complete the classification. (2) This paper proposes to use the frequency domain spatial interpolation method for efficient alignment in each set of data after the classification is completed, and then complete the class average calculation. ...
Background
Cryo-electron microscopy (Cryo-EM) plays an increasingly important role in the determination of the three-dimensional (3D) structure of macromolecules. In order to achieve 3D reconstruction results close to atomic resolution, 2D single-particle image classification is not only conducive to single-particle selection, but also a key step that affects 3D reconstruction. The main task is to cluster and align 2D single-grain images into non-heterogeneous groups to obtain sharper single-grain images by averaging calculations. The main difficulties are that the cryo-EM single-particle image has a low signal-to-noise ratio (SNR), cannot manually label the data, and the projection direction is random and the distribution is unknown. Therefore, in the low SNR scenario, how to obtain the characteristic information of the effective particles, improve the clustering accuracy, and thus improve the reconstruction accuracy, is a key problem in the 2D image analysis of single particles of cryo-EM.
Results
Aiming at the above problems, we propose a learnable deep clustering method and a fast alignment weighted averaging method based on frequency domain space to effectively improve the class averaging results and improve the reconstruction accuracy. In particular, it is very prominent in the feature extraction and dimensionality reduction module. Compared with the classification method based on Bayesian and great likelihood, a large amount of single particle data is required to estimate the relative angle orientation of macromolecular single particles in the 3D structure, and we propose that the clustering method shows good results.
Conclusions
SimcryoCluster can use the contrastive learning method to perform well in the unlabeled high-noise cryo-EM single particle image classification task, making it an important tool for cryo-EM protein structure determination
... The full 1 H-13 C HSQC spectrum indicated that neither proteins nor polysaccharides were present in the NA gel isolate (Supplementary Fig. 1) 21 . eDNA fibre networked structures were visible in the polysaccharide mutants and the proteinase K digested PAO1 wildtype biofilms ( Supplementary Fig. 2a-c). ...
The extracellular matrix of bacterial biofilms consists of diverse components including polysaccharides, proteins and DNA. Extracellular RNA (eRNA) can also be present, contributing to the structural integrity of biofilms. However, technical difficulties related to the low stability of RNA make it difficult to understand the precise roles of eRNA in biofilms. Here, we show that eRNA associates with extracellular DNA (eDNA) to form matrix fibres in Pseudomonas aeruginosa biofilms, and the eRNA is enriched in certain bacterial RNA transcripts. Degradation of eRNA associated with eDNA led to a loss of eDNA fibres and biofilm viscoelasticity. Compared with planktonic and biofilm cells, the biofilm matrix was enriched in specific mRNA transcripts, including lasB (encoding elastase). The mRNA transcripts colocalised with eDNA fibres in the biofilm matrix, as shown by single molecule inexpensive FISH microscopy (smiFISH). The lasB mRNA was also observed in eDNA fibres in a clinical sputum sample positive for P. aeruginosa. Thus, our results indicate that the interaction of specific mRNAs with eDNA facilitates the formation of viscoelastic networks in the matrix of Pseudomonas aeruginosa biofilms.
... DSS was used as a chemical shift standard, and 15 N and 13 C data were referenced using frequency ratios [55]. Peak assignment was performed using the program SPARKY [56] and standard methods [57]. 15 N HSQC spectrum of the final titration step was assigned using the 3D spectrum, programs and methods described for pep3. ...
Poly‐proline II helices are secondary structure motifs frequently found in ligand‐binding sites. They exhibit increased flexibility and solvent exposure compared to the strongly hydrogen‐bonded α‐helices or β‐strands and can therefore easily be misinterpreted as completely unstructured regions with an extremely high rotational freedom. Here, we show that the adhesin YadA of Yersinia enterocolitica serotype O:9 contains a poly‐proline II helix interaction motif in the N‐terminal region. The motif is involved in the interaction of YadAO:9 with heparin, a host glycosaminoglycan. We show that the basic residues within the N‐terminal motif of YadA are required for electrostatic interactions with the sulfate groups of heparin. Biophysical methods including CD spectroscopy, solution‐state NMR and SAXS all independently support the presence of a poly‐proline helix allowing YadAO:9 binding to the rigid heparin. Lastly, we show that host cells deficient in sulfation of heparin and heparan sulfate are not targeted by YadAO:9‐mediated adhesion. We speculate that the YadAO:9–heparin interaction plays an important and highly strain‐specific role in the pathogenicity of Yersinia enterocolitica serotype O:9.
... Third, we recorded and analyzed nuclear Overhauser effect spectra (NOESY), which allowed us to detect pairs of 1 H atoms lying in close spatial proximity (typically <5 Å) (Wüthrich 1986). We successfully assigned a number of side chain methyl and aromatic groups of both Rec114 and Mei4, which are buried in the core of the protein complex and thus can be used to detect specific residue-residue contacts. ...
Meiosis-specific Rec114−Mei4 and Mer2 complexes are thought to enable Spo11-mediated DNA double-strand break (DSB) formation through a mechanism that involves DNA-dependent condensation. However, the structure, molecular properties, and evolutionary conservation of Rec114−Mei4 and Mer2 are unclear. Here, we present AlphaFold models of Rec114−Mei4 and Mer2 complexes supported by nuclear magnetic resonance (NMR) spectroscopy, small-angle X-ray scattering (SAXS), and mutagenesis. We show that dimers composed of the Rec114 C terminus form α-helical chains that cup an N-terminal Mei4 α helix, and that Mer2 forms a parallel homotetrameric coiled coil. Both Rec114−Mei4 and Mer2 bind preferentially to branched DNA substrates, indicative of multivalent protein–DNA interactions. Indeed, the Rec114−Mei4 interaction domain contains two DNA-binding sites that point in opposite directions and drive condensation. The Mer2 coiled-coil domain bridges coaligned DNA duplexes, likely through extensive electrostatic interactions along the length of the coiled coil. Finally, we show that the structures of Rec114−Mei4 and Mer2 are conserved across eukaryotes, while DNA-binding properties vary significantly. This work provides insights into the mechanism whereby Rec114−Mei4 and Mer2 complexes promote the assembly of the meiotic DSB machinery and suggests a model in which Mer2 condensation is the essential driver of assembly, with the DNA-binding activity of Rec114−Mei4 playing a supportive role.
... Biophysical characterization of antigenic peptides by nuclear magnetic resonance (NMR) spectroscopy NMR measurements were performed at 2 mM peptide in a DMSO-d6 solution. NMR experiments were recorded using a Bruker Avance III HD 800 MHz spectrometer (Federal University of Rio de Janeiro, Rio de Janeiro, Brazil) at 300 K. Two-dimensional experiments were recorded for sequential NMR assignment (57) and distance restriction derivation. These included 1 H-1 H total correlation spectroscopy using the decoupling in the presence of scalar interactions-2 pulse sequence (58) with water suppression. ...
COVID-19 has accounted for more than 6 million deaths worldwide. Bacillus Calmette–Guérin (BCG), the existing tuberculosis vaccine, is known to induce heterologous effects over other infections due to trained immunity and has been proposed to be a potential strategy against SARS-CoV-2 infection. In this report, we constructed a recombinant BCG (rBCG) expressing domains of the SARS-CoV-2 nucleocapsid and spike proteins (termed rBCG-ChD6), recognized as major candidates for vaccine development. We investigated whether rBCG-ChD6 immunization followed by a boost with the recombinant nucleocapsid and spike chimera (rChimera), together with alum, provided protection against SARS-CoV-2 infection in K18-hACE2 mice. A single dose of rBCG-ChD6 boosted with rChimera associated with alum elicited the highest anti-Chimera total IgG and IgG2c Ab titers with neutralizing activity against SARS-CoV-2 Wuhan strain when compared with control groups. Importantly, following SARS-CoV-2 challenge, this vaccination regimen induced IFN-γ and IL-6 production in spleen cells and reduced viral load in the lungs. In addition, no viable virus was detected in mice immunized with rBCG-ChD6 boosted with rChimera, which was associated with decreased lung pathology when compared with BCG WT-rChimera/alum or rChimera/alum control groups. Overall, our study demonstrates the potential of a prime-boost immunization system based on an rBCG expressing a chimeric protein derived from SARS-CoV-2 to protect mice against viral challenge.
Alamethicin, a peptide consisted of 20 amino acid residues, has been known to function as an antibiotic. The peptides self-associate in biological membranes, form an ion channel, and then induce cell death by leaking intracellular contents through a transmembrane pore of an ion channel. We investigated conformation and its thermal stability of alamethicin-A6 and -U6 in ethanol using proton nuclear magnetic resonance (NMR) spectroscopy; alamethicin-A6 and -U6 have the amino acid sequences of UPUAUAQUVUGLUPVUUQQO and UPUAUUQUVUGLUPVUUQQO, respectively, where U and O represent α-aminoisobutyric acid and phenylalaninol, respectively. As indicated by the under bars in the sequences, only the residue 6 differs between the alamethicins. We show that the alamethicins in ethanol form helix conformation in the region of the residues 2–11 and a non-regular conformation in the regions of the N- and C-termini, and that the helices are maintained up to 66 °C at least. Conformations in the region of the residues 12–18 of the alamethicins, however, are not well identified due to the lack of NMR data. In addition, we demonstrate that the amide proton chemical shift temperature coefficients’ method, which is known as an indicator for intramolecular hydrogen bonds in peptides and proteins in aqueous solutions, can be also applied to the alamethicins in ethanol. Further, we show that the conformation around the C-terminus of alamethicin-A6 is restrained by intramolecular hydrogen bonds, whereas that of alamethicin-U6 is either restrained or unrestrained by intramolecular hydrogen bonds; the alamethicin-U6 molecules having the restrained and unrestrained conformations coexist in ethanol. We discuss the two types of conformations using a model chain consisting of particles linked by rigid bonds called as the free jointed chain.
Molecular dynamics simulations depend critically on the quality of the force field used to describe the interatomic interactions and the extent to which it has been validated for use in a specific application. Using a curated test set of 52 high-resolution structures, 39 derived from X-ray diffraction and 13 solved using NMR, we consider the extent to which different parameter sets of the GROMOS protein force field can be distinguished based on comparing a range of structural criteria, including the number of backbone hydrogen bonds, the number of native hydrogen bonds, polar and nonpolar solvent-accessible surface area, radius of gyration, the prevalence of secondary structure elements, J-coupling constants, nuclear Overhauser effect (NOE) intensities, positional root-mean-square deviations (RMSD), and the distribution of backbone ϕ and ψ dihedral angles. It is shown that while statistically significant differences between the average values of individual metrics could be detected, these were in general small. Furthermore, improvements in agreement in one metric were often offset by loss of agreement in another. The work establishes a framework and test set against which protein force fields can be validated. It also highlights the danger of inferring the relative quality of a given force field based on a small range of structural properties or small number of proteins.
The development of turn-based inhibitors of protein–protein interactions has attracted considerable attention in medicinal chemistry. Our group has synthesized a series of peptides derived from an amino-functionalized ferrocene to investigate their potential to mimic protein turn structures. Detailed DFT and spectroscopic studies (IR, NMR, CD) have shown that, for peptides, the backbone chirality and bulkiness of the amino acid side chains determine the hydrogen-bond pattern, allowing tuning of the size of the preferred hydrogen-bonded ring in turn-folded structures. However, their biological potential is more dependent on their lipophilicity. In addition, our pioneering work on the chiroptical properties of aminoferrocene-containing peptides enables the correlation of their geometry with the sign of the CD signal in the absorption region of the ferrocene chromophore. These studies have opened up the possibility of using aminoferrocene and its derivatives as chirooptical probes for the determination of various chirality elements, such as the central chirality of amino acids and the helicity of peptide sequences.
Peptide dhvar4, derived from the active domain of our salivary peptide histatin 5, bears a Phe residue in the middle of its hydrophilic face when folded into an α‐helix. We then synthesized an analog with this Phe replaced by Lys and two analogs preserving Phe but bearing two and three α‐aminoisobutyric acid (Aib) residues to stabilize the helical structure. The aim of this design was to verify which of the two features is more favorable to the biological activity. We performed a conformational study by means of circular dichroism and nuclear magnetic resonance, made antibacterial tests, and assessed the stability of the peptides in human serum. We observed that amphiphilicity is more important than helix stability, provided a peptide can adopt a helical conformation in a membrane‐mimetic environment.
Protein–protein interaction is at the heart of most biological processes, and small peptides that bind to protein binding sites are resourceful tools to explore and understand the structural requirements for these interactions. In that sense, phage display is a well-suited technology to study protein–protein interactions, as it allows for unbiased screening of billions of peptides in search for those that interact with a protein binding domain. Here, we will illustrate how two distinct but complementary approaches, phage display and nuclear magnetic resonance (NMR), can be utilized to unveil structural details of peptide–protein interaction. Finally, knowledge derived from phage mutagenesis and NMR studies can be streamlined for quick peptidomimetic design and synthesis using the retroinversion approach to validate using in vitro and in vivo assays the therapeutic potential of peptides identified by phage display.
Annexin A11 (ANXA11) is a calcium-dependent phospholipid-binding protein belonging to the annexin protein family and implicated in the neurodegenerative amyotrophic lateral sclerosis. Structurally, ANXA11 contains a conserved calcium-binding C-terminal domain common to all annexins and a putative intrinsically unfolded N-terminus specific for ANXA11. Little is known about the structure and functions of this region of the protein. By analogy with annexin A1, it was suggested that residues 38 to 59 within the ANXA11 N-terminus could form a helical region that would be involved in interactions. Interestingly, this region contains residues that, when mutated, may lead to clinical manifestations. In the present study, we have studied the structural features of the full-length protein with special attention to the N-terminal region using a combination of biophysical techniques which include nuclear magnetic resonance and small angle X-ray scattering. We show that the N-terminus is intrinsically disordered and that the overall features of the protein are not markedly affected by the presence of calcium. We also analyzed the 38–59 helix hypothesis using synthetic peptides spanning both the wild-type sequence and clinically relevant mutations. We show that the peptides have a remarkable character typical of a native helix and that mutations do not alter the behaviour suggesting that they are required for interactions rather than being structurally important. Our work paves the way to a more thorough understanding of the ANXA11 functions.
A series of 10 cyclic, biaryl analogs of enkephalin, with Tyr or Phe residues at positions 1 and 4, were synthesized according to the Miyaura borylation and Suzuki coupling methodology. Biaryl bridges formed by side chains of the two aromatic amino acid residues are of the meta–meta, meta–para, para–meta, and para–para configuration. Conformational properties of the peptides were studied by CD and NMR. CD studies allowed only to compare conformations of individual peptides while NMR investigations followed by XPLOR calculations provided detailed information on their conformation. Reliability of the XPLOR calculations was confirmed by quantum chemical ones performed for one of the analogs. No intramolecular hydrogen bonds were found in all the peptides. They are folded and adopt the type IV β-turn conformation. Due to a large steric strain, the aromatic carbon atoms forming the biaryl bond are distinctly pyramidalized. Seven of the peptides were tested in vitro for their affinity for the µ-opioid receptor.
Supplementary Information
The online version contains supplementary material available at 10.1007/s00726-023-03371-5.
Residual dipolar couplings (RDCs) are NMR measurements widely used to determine structural and dynamic information in small molecules and large macromolecules. This book provides a broad view of RDCs, from basic principles to advanced applications in organic molecules and biomolecules. Exploring the newest developments in RDC measurement and analysis through authoritative accounts written by leaders in the field, this book provides a comprehensive overview on the fundamentals, analysis and applications in one place for the first time.
The versatility and accuracy of RDCs have found a large range of applications in NMR, and their measurement and analysis are major research areas. Readers, be they experts or students, will receive a strong understanding of the fundamentals of RDCs and their applications to their research projects.
The difficulty in evaluating the conformational distribution of proteins in solution often hinders mechanistic insights. One possible strategy for visualizing conformational distribution is distance distribution measurement by single-pair small-angle X-ray scattering (SAXS), in which the scattering interference from only a specific pair of atoms in the target molecule is extracted. Despite this promising concept, with few applications in synthetic small molecules and DNA, technical difficulties have prevented its application in protein conformational studies. This study used a synthetic tag to fix the lanthanide ion at desired sites on the protein and used single-pair SAXS with contrast matching to evaluate the conformational distribution of the multidomain protein enzyme MurD. These data highlighted the broad conformational and ligand-driven distribution shifts of MurD in solution. This study proposes an important strategy in solution structural biology that targets dynamic proteins, including multidomain and intrinsically disordered proteins.
Nuclear Magnetic Resonance (NMR) spectroscopy is the ideal tool to address the structure, reactivity and dynamics of both inorganic and biological substances. The knowledge of nuclear spin interaction and spin dynamics is increasingly consolidated, and this allows for tailoring pulse sequences. When dealing with paramagnetic systems, several decades of research have led to the development of rule-of-the-thumb criteria for optimizing the experiments, allowing for the detection of nuclei that are in very close proximity to the metal center. In turn, the observation of these systems, coupled with the development of robust and accessible quantum chemical methods, is promising to provide a link between the spectra and the structural features through the interpretation of the electronic structure. In this review, we list the challenges encountered and propose solutions for dealing with paramagnetic systems with the greatest satisfaction. In our intentions, this is a practical toolkit for optimizing acquisition and processing parameters for routine experiments aimed at detecting signals influenced by the hyperfine interaction. The implications of paramagnetic shift and line broadening are examined. With this endeavor, we wish to encourage non-expert users to consider the application of paramagnetic NMR to their systems.
Modern research in biology increasingly relies on multiple techniques for describing structures and mechanisms. This book provides an overview of the contemporary integrated biology approaches for solving structures and understanding mechanisms of complex biological systems. It includes several methodology chapters discussing the current developments in the areas of cryo- electron microscopy (EM) and cryo-electron tomography (ET), computational biophysics, solution NMR spectroscopy, solid-state NMR spectroscopy and dynamic nuclear polarization (DNP), electron paramagnetic resonance (EPR), (photo-)chemically induced dynamic nuclear polarization (CIDNP), X-ray crystallography and small-angle X-ray and neutron scattering (SAXS/SANS). Several subsequent chapters demonstrate how these methods are used in synergy to address problems at the forefront of structural biology, with particular emphasis on examples where individual techniques are insufficient. Examples of biological systems include membrane proteins, viral protein assemblies, cytoskeleton protein assemblies, photosynthetic reaction centers, large enzyme complexes and whole cells. The book is targeted to both the current practitioners of structural biology and scientists who are interested in entering the fields of structural biology or biophysical chemistry.
2D NOESY and TOCSY play central roles in contemporary NMR. We have recently discussed how solvent-driven exchanges can significantly enhance the sensitivity of such methods when attempting correlations between labile and nonlabile protons. This study explores two scenarios where similar sensitivity enhancements can be achieved in the absence of solvent exchange: the first one involves biomolecular paramagnetic systems, while the other involves small organic molecules in natural abundance. It is shown that, in both cases, the effects introduced by either differential paramagnetic shift and relaxation or by polarization sharing among networks of protons can provide a similar sensitivity boost, as previously discussed for solvent exchange. The origin and potential of the resulting enhancements are analyzed, and experiments that demonstrate them in protein and natural products are exemplified. Limitations and future improvements of these approaches are also briefly discussed.
Chemical shift assignment is vital for nuclear magnetic resonance (NMR)–based studies of protein structures, dynamics, and interactions, providing crucial atomic-level insight. However, obtaining chemical shift assignments is labor intensive and requires extensive measurement time. To address this limitation, we previously proposed ARTINA, a deep learning method for automatic assignment of two-dimensional (2D)–4D NMR spectra. Here, we present an integrative approach that combines ARTINA with AlphaFold and UCBShift, enabling chemical shift assignment with reduced experimental data, increased accuracy, and enhanced robustness for larger systems, as presented in a comprehensive study with more than 5000 automated assignment calculations on 89 proteins. We demonstrate that five 3D spectra yield more accurate assignments (92.59%) than pure ARTINA runs using all experimentally available NMR data (on average 10 3D spectra per protein, 91.37%), considerably reducing the required measurement time. We also showcase automated assignments of only ¹⁵ N-labeled samples, and report improved assignment accuracy in larger synthetic systems of up to 500 residues.
Bacterial secretion systems are built up from proteins with different physicochemical characteristics, such as highly hydrophobic transmembrane polypeptides, and soluble periplasmic or intracellular domains. A single complex can be composed of more than ten proteins with distinct features, spreading through different cellular compartments. The membrane and multicompartment nature of the proteins, and their large molecular weight make their study challenging. However, information on their structure and assemblies is required to understand their mechanisms and interfere with them. An alternative strategy is to work with soluble domains and peptides corresponding to the regions of interest of the proteins.
Here, we describe a simple and fast protocol to evaluate the stability, folding, and interaction of protein sub-complexes by using solution-state Nuclear Magnetic Resonance (NMR) spectroscopy. This technique is widely used for protein structure and protein–ligand interaction analysis in solution.
Simple Summary
Glioblastoma is the most devastating and widespread primary central nervous system tumor and there is a compelling need for innovative and effective therapeutic strategies. The remarkable vascularization sustaining this malignancy occurs through different mechanisms, including vasculogenic mimicry, i.e., the non-endothelial formation of new vessels, that is a promising therapeutic target. This study provides evidence that the recently described anti-invasive uPAcyclin decapeptide is remarkably active in reducing migration, invasion, and vasculogenic mimicry formation by human glioblastoma cells. These findings uncover novel uPAcyclin activities and provide a strong rationale for further clinical studies.
Abstract
Among the deadliest human cancers is glioblastoma (GBM) for which new treatment approaches are urgently needed. Here, the effects of the cyclic decapeptide, uPAcyclin, are investigated using the U87-MG, U251-MG, and U138-MG human GBM and C6 rat cell models. All GBM cells express the αV-integrin subunit, the target of uPAcyclin, and bind specifically to nanomolar concentrations of the decapeptide. Although peptide exposure affects neither viability nor cell proliferation rate, nanomolar concentrations of uPAcyclin markedly inhibit the directional migration and matrix invasion of all GBM cells, in a concentration- and αV-dependent manner. Moreover, wound healing rate closure of U87-MG and C6 rat glioma cells is reduced by 50% and time-lapse videomicroscopy studies show that the formation of vascular-like structures by U87-MG in three-dimensional matrix cultures is markedly inhibited by uPAcyclin. A strong reduction in the branching point numbers of the U87-MG, C6, and U251-MG cell lines undergoing vasculogenic mimicry, in the presence of nanomolar peptide concentrations, was observed. Lysates from matrix-recovered uPAcyclin-exposed cells exhibit a reduced expression of VE-cadherin, a prominent factor in the acquisition of vascular-like structures. In conclusion, these results indicate that uPAcyclin is a promising candidate to counteract the formation of new vessels in novel targeted anti-GBM therapies.
The matrix pencil method (MPM) is a powerful tool for processing transient nuclear magnetic resonance (NMR) relaxation signals with promising applications to increasingly complex problems. In the absence of signal noise, the eigenvalues recovered from an MPM treatment of transient relaxometry data reduce to relaxation coefficients that can be used to calculate relaxation time constants for known sampling time ∆t. The MPM eigenvalue and relaxation coefficient equality as well as the resolution of similar eigenvalues and thus relaxation coefficients degrade in the presence of signal noise. The relaxation coefficient ∆t dependence suggests one way to improve MPM resolution by choosing ∆t values such that the differences between all the relaxation coefficient values are maximized. This work develops mathematical machinery to estimate the best ∆t value for sampling damped, transient relaxation signals such that MPM data analysis recovers a maximum number of time constants and amplitudes given inherent signal noise. Analytical and numerical reduced dimension MPM is explained and used to compare computer-generated data with and without added noise as well as treat real measured signals. Finally, the understanding gleaned from this effort is used to predict the best data sampling time to use for non-discrete, distributions of relaxation variables.
Cathelicidins, a family of host defence peptides in vertebrates, play an important role in the innate immune response, exhibiting antimicrobial activity against many bacteria, as well as viruses and fungi. This work describes the design and synthesis of shortened analogues of porcine cathelicidin PMAP-36, which contain structural changes to improve the pharmacokinetic properties. In particular, 20-mers based on PMAP-36 (residues 12-31) and 13-mers (residues 12-24) with modification of amino acid residues at critical positions and introduction of lipid moieties of different lengths were studied to identify the physical parameters, including hydrophobicity, charge, and helical structure, required to optimise their antibacterial activity. Extensive conformational analysis, performed by CD and NMR, revealed that the substitution of Pro25-Pro26 with Ala25-Lys26 increased the α-helix content of the 20-mer peptides, resulting in broad-spectrum antibacterial activity against Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Staphylococcus epidermidis strains. Interestingly, shortening to just 13 residues resulted in only a slight decrease in antibacterial activity. Furthermore, two sequences, a 13-mer and a 20-mer, did not show cytotoxicity against HaCat cells up to 64 µM, indicating that both derivatives are not only effective but also selective antimicrobial peptides. In the short peptide, the introduction of the helicogenic α-aminoisobutyric acid forced the helix toward a prevailing 3 10 structure, allowing the antimicrobial activity to be maintained. Preliminary tests of resistance to Ser protease chymotrypsin indicated that this modification resulted in a peptide with an increased in vivo lifespan. Thus, some of the PMAP-36 derivatives studied in this work show a good balance between chain length, antibacterial activity, and selectivity, so they represent a good starting point for the development of even more effective and proteolysis-resistant active peptides.
T:G mismatches in mammals arise primarily from the deamination of methylated CpG sites or the incorporation of improper nucleotides. The process by which repair enzymes such as thymine DNA glycosylase (TDG) identify a canonical DNA base in the incorrect pairing context remains a mystery. However, the abundant contacts of the repair enzymes with the DNA backbone suggest a role for protein–phosphate interaction in the recognition and repair processes, where conformational properties may facilitate the proper interactions. We have previously used ³¹P NMR to investigate the energetics of DNA backbone BI–BII interconversion and the effect of a mismatch or lesion compared to canonical DNA and found stepwise differences in ΔG of 1–2 kcal/mol greater than equivalent steps in unmodified DNA. We have currently compared our results to substrate dependence for TDG, MBD4, M. HhaI, and CEBPβ, testing for correlations to sequence and base-pair dependence. We found strong correlations of our DNA phosphate backbone equilibrium (Keq) to different enzyme kinetics or binding parameters of these varied enzymes, suggesting that the backbone equilibrium may play an important role in mismatch recognition and/or conformational rearrangement and energetics during nucleotide flipping or other aspects of enzyme interrogation of the DNA substrate.
Sunflower trypsin inhibitor‐1 (SFTI‐1) structure is used for designing‐grafted peptides as a possible therapeutic agent. The grafted peptide exhibits multiple conformations in solution due to the presence of proline in the structure of the peptide. To lock the grafted peptide into a major conformation in solution, a dibenzofuran moiety (DBF) was incorporated in the peptide backbone structure, replacing the Pro‐Pro sequence. NMR studies indicated a major conformation of the grafted peptide in solution. Detailed structural studies suggested that SFTI‐DBF adopts a twisted beta‐strand structure in solution. The surface plasmon resonance analysis showed that SFTI‐DBF binds to CD58 protein. A model for the protein‐SFTI‐DBF complex was proposed based on docking studies. These studies suggested that SFTI‐1 grafted peptide can be used to design stable peptides for therapeutic purposes by grafting organic functional groups and amino acids. However, when a similar strategy was used with another grafted peptide, the resulting peptide did not produce a single major conformation, and its biological activity was lost. Thus, conformational constraints depend on the sequence of amino acids used for SFTI‐1 grafting.
Eight hundred and twenty-six human G protein-coupled receptors (GPCRs) mediate the actions of two-thirds of the human hormones and neurotransmitters and over one-third of clinically used drugs. Studying the structure and dynamics of human GPCRs in lipid bilayer environments resembling the native cell membrane milieu is of great interest as a basis for understanding structure–function relationships and thus benefits continued drug development. Here, we incorporate the human A2A adenosine receptor (A2AAR) into lipid nanodiscs, which represent a detergent-free environment for structural studies using nuclear magnetic resonance (NMR) in solution. The [15N,1H]-TROSY correlation spectra confirmed that the complex of [u-15N, ~70% 2H]-A2AAR with an inverse agonist adopts its global fold in lipid nanodiscs in solution at physiological temperature. The global assessment led to two observations of practical interest. First, A2AAR in nanodiscs can be stored for at least one month at 4 °C in an aqueous solvent. Second, LMNG/CHS micelles are a very close mimic of the environment of A2AAR in nanodiscs. The NMR signal of five individually assigned tryptophan indole 15N–1H moieties located in different regions of the receptor structure further enabled a detailed assessment of the impact of nanodiscs and LMNG/CHS micelles on the local structure and dynamics of A2AAR. As expected, the largest effects were observed near the lipid–water interface along the intra- and extracellular surfaces, indicating possible roles of tryptophan side chains in stabilizing GPCRs in lipid bilayer membranes.
The binding affinity of G protein-coupled receptor (GPCR) ligands is customarily measured by radio-ligand competition experiments. As an alternative approach, 19F nuclear magnetic resonance spectroscopy (19F-NMR) is used for the screening of small-molecule lead compounds in drug discovery; the two methods are complementary in that the measurements are performed with widely different experimental conditions. Here, we used the structure of the A2A adenosine receptor (A2AAR) complex with V-2006 (3-(4-amino-3-methylbenzyl)-7-(furan-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine) as the basis for the design of a fluorine-containing probe molecule, FPPA (4-(furan-2-yl)-7-(4-(trifluoromethyl)benzyl)-7H-pyrrolo[2,3-d]pyramidin-2-amine), for binding studies with A2AAR. A protocol of experimental conditions for drug screening and measurements of drug binding affinities using 1D 19F-NMR observation of FPPA is validated with studies of known A2AAR ligands. 19F-NMR with FPPA is thus found to be a robust approach for the discovery of ligands with new core structures, which will expand the libraries of A2AAR-targeting drug candidates.
The effect of attaching the achiral, cyclic 1-aminocyclohexanecarboxylic acid (Ac6c) directly to the aminoferrocene unit (Ac6c−NH−Fc) appears to be a promising route for the development of a new chiroptical sensor based on a ferrocene chromophore. Three new compounds (Boc−AA−Ac6c−NH−Fc; AA = L-Ala, L-Val, L-Phe) were synthesized, spectroscopically characterized (IR, NMR, CD), and conformationally analyzed (DFT). The chiral information was transferred from the L-amino acid to the ferrocene chromophore by the predominant formation of P-helical structures with ten-membered hydrogen-bonded rings (β-turns). The perturbation of the ferrocene chromophore and the appearance of the negative CD signal near 470 nm originates from a relative orientation of the directly linked amide and cyclopentadienyl planes, described by the dihedral angle χ. The sterically demanding Ac6c amino acid makes trans-like configurations more favorable and thus restricts the dihedral angle χ, which then leads to the appearance of the negative peak near 470 nm in the CD curve.
The stability of cyclic peptides, coupled with their structural diversity and ability to host an extensive range of bioactivities, make them promising leads for the development of new drugs. PawS-Derived Peptide-23 (PDP-23) is a head-to-tail macrocyclic peptide with two disulfide bonds produced in plant seeds. Its unusual fold comprises two -hairpins connected by hinges that allow the structure to adapt to different environments. In water two PDP-23 molecules form a compact intertwined dimer that buries hydrophobic residues, whereas in membrane mimicking conditions it adopts an open monomeric form that expose them. Here we investigate PDP-23 as a novel scaffold for the grafting of bioactive epitopes and conjugation of small molecules. To explore the plasticity of PDP-23 we introduced the bioactive loop of sunflower trypsin inhbitor-1 (SFTI-1) or an integrin binding RGD motif into either of the -hairpins. Solution NMR spectroscopy revealed that although the variants were unable to dimerise, the structural features of both the graft and scaffold were retained. SFTI-1 hybrid variants showed trypsin inhibitory activity. PDP-23 has previously been used as a cell permeable drug scaffold targeting drug-resistant cancer cells by inhibiting the drug efflux pump P-glycoprotein and restoring their sensitivity to chemotherapeutic. Introducing the RGD motif into such PDP-23 conjugates significantly improve their potency, suggesting that the RGD sequence targets the peptide to the membrane of cancer cells and improve cell uptake. In conclusion, this study highlights PDP-23 as a stable and versatile scaffold for molecular grafting of bioactivities and targeted delivery of pharmaceutical payloads.
Lantibiotics are ribosomally synthesized and posttranslationally modified peptides (RiPPs) that are produced by bacteria. Interest in this group of natural products is increasing rapidly as alternatives to conventional antibiotics. Some human microbiome-derived commensals produce lantibiotics to impair pathogens' colonization and promote healthy microbiomes. Streptococcus salivarius is one of the first commensal microbes to colonize the human oral cavity and gastrointestinal tract, and its biosynthesis of RiPPs, called salivaricins, has been shown to inhibit the growth of oral pathogens. Herein, we report on a phosphorylated class of three related RiPPs, collectively referred to as salivaricin 10, that exhibit proimmune activity and targeted antimicrobial properties against known oral pathogens and multispecies biofilms. Strikingly, the immunomodulatory activities observed include upregulation of neutrophil-mediated phagocytosis, promotion of antiinflammatory M2 macrophage polarization, and stimulation of neutrophil chemotaxis-these activities have been attributed to the phosphorylation site identified on the N-terminal region of the peptides. Salivaricin 10 peptides were determined to be produced by S. salivarius strains found in healthy human subjects, and their dual bactericidal/antibiofilm and immunoregulatory activity may provide new means to effectively target infectious pathogens while maintaining important oral microbiota.
It is known that four peptide fragments of predominant protein in human semen Semenogelin 1 (SEM1) (SEM1(86–107), SEM1(68–107), SEM1(49–107) and SEM1(45–107)) are involved in fertilization and amyloid formation processes. In this work, the structure and dynamic behavior of SEM1(45–107) and SEM1(49–107) peptides and their N-domains were described. According to ThT fluorescence spectroscopy data, it was shown that the amyloid formation of SEM1(45–107) starts immediately after purification, which is not observed for SEM1(49–107). Seeing that the peptide amino acid sequence of SEM1(45–107) differs from SEM1(49–107) only by the presence of four additional amino acid residues in the N domain, these domains of both peptides were obtained via solid-phase synthesis and the difference in their dynamics and structure was investigated. SEM1(45–67) and SEM1(49–67) showed no principal difference in dynamic behavior in water solution. Furthermore, we obtained mostly disordered structures of SEM1(45–67) and SEM1(49–67). However, SEM1(45–67) contains a helix (E58-K60) and helix-like (S49-Q51) fragments. These helical fragments may rearrange into β-strands during amyloid formation process. Thus, the difference in full-length peptides’ (SEM1(45–107) and SEM1(49–107)) amyloid-forming behavior may be explained by the presence of a structured helix at the SEM1(45–107) N-terminus, which contributes to an increased rate of amyloid formation.
Nuclear magnetic resonance (NMR) is a paramount analytical tool for chemistry, biology, medicine, and geology, and has a fundamental importance in physics. The recent years have seen a wealth of efforts to miniaturize NMR systems by combining permanent magnets and CMOS radio frequency (RF)-integrated circuits (ICs) to make the benefit of NMR more broadly available beyond dedicated facilities, which have resulted in systems capable of NMR relaxometry first, and later NMR spectroscopy, the two key NMR modalities. Here we report a small NMR system comprising a digitally assisted CMOS RF transceiver IC and a 0.51-T permanent magnet, which not only enhances spectroscopy and relaxometry performance but also includes magnetic resonance imaging (MRI), a powerful variant of relaxometry. The system achieves a spectral resolution of
$<$
0.05 ppm (1.1 Hz), the highest reported in a portable CMOS-based NMR system, and an imaging resolution of
$67\times 67$
$\times$
83
$\bm{\mu}$
m
$^3$
.
This chapter focuses on analytical procedures such as extraction, isolation, and characterization of phytoactive components. The application of conventional phytochemical screening assays, chromatographic techniques such as HPLC and TLC, as well as non-chromatographic techniques such as immunoassay, Fourier transform infrared (FTIR), GCMS, enzymatic extraction, ultrasonic extraction, and other analytical techniques developed recently to the detection of bioactive chemicals present in plant extracts is reviewed. The aim of this chapter is to gain a deep understanding of the fruitful achievements and to provide perspectives, trends, and directions regarding further research in novel extraction and characterization methods for phytochemicals. These efforts have led to the discovery of various important clinical drugs.
This chapter also focuses on analytical techniques like phytoactive component extraction, isolation, and characterization. The use of traditional phytochemical screening assays, chromatographic methods like HPLC and TLC, as well as non-chromatographic methods like immunoassay, FTIR, GCMS, enzymatic extraction, ultrasonic extraction, and other analytical methods developed recently is reviewed. This chapter’s goals are to present viewpoints, trends, and directions for future research on novel extraction and characterization techniques for phytochemicals as well as a thorough knowledge of the successful accomplishments.
NMR isotope shifts occur due to small differences in nuclear shielding when nearby atoms are different isotopes. For molecules dissolved in 1:1 H2O:D2O, the resulting mixture of N-H and N-D isotopes leads to a small splitting of resonances from adjacent nuclei. We used multidimensional NMR to measure isotope shifts for the proteins CUS-3iD and CspA. We observed four-bond ⁴∆N(ND) isotope shifts in high-resolution 2D ¹⁵N-TROSY experiments of the perdeuterated proteins that correlate with the torsional angle psi. Three-bond ³∆C’(ND) isotope shifts detected in H(N)CO spectra correlate with the intraresidue H-O distance, and to a lesser extent with the dihedral angle phi. The conformational dependence of the isotope shifts agree with those previously reported in the literature. Both the ⁴∆N(ND) and ³∆C’(ND) isotope shifts are sensitive to distances between the atoms giving rise to the isotope shifts and the atoms experiencing the splitting, however, these distances are strongly correlated with backbone dihedral angles making it difficult to resolve distance from stereochemical contributions to the isotope shift. H(NCA)CO spectra were used to measure two-bond ²∆C’(ND) isotope shifts and [D]/[H] fractionation factors. Neither parameter showed significant differences for hydrogen-bonded sites, or changes over a 25° temperature range, suggesting they are not sensitive to hydrogen bonding. Finally, the quartet that arises from the combination of ²∆C’(ND) and ³∆C’(ND) isotope shifts in H(CA)CO spectra was used to measure synchronized hydrogen exchange for the sequence neighbors A315-S316 in the protein CUS-3iD. In many of our experiments we observed minor resonances due to the 10% D2O used for the sample deuterium lock, indicating isotope shifts can be a source of spectral heterogeneity in standard NMR experiments. We suggest that applications of isotope shifts such as conformational analysis and correlated hydrogen exchange could benefit from the larger magnetic fields becoming available.
ResearchGate has not been able to resolve any references for this publication.