An Upper Limit for Macromolecular Crowding Effects

Abstract

Solutions containing high macromolecule concentrations are predicted to affect a number of protein properties compared to those properties in dilute solution. In cells, these macromolecular crowders have a large range of sizes and can occupy 30% or more of the available volume. We chose to study the stability and ps-ns internal dynamics of a globular protein whose radius is ~2 nm when crowded by a synthetic microgel composed of poly(N-isopropylacrylamide-co-acrylic acid) with particle radii of ~300 nm.
Our studies revealed no change in protein rotational or ps-ns backbone dynamics and only mild (~0.5 kcal/mol at 37°C, pH 5.4) stabilization at a volume occupancy of 70%, which approaches the occupancy of closely packing spheres. The lack of change in rotational dynamics indicates the absence of strong crowder-protein interactions.
Our observations are explained by the large size discrepancy between the protein and crowders and by the internal structure of the microgels, which provide interstitial spaces and internal pores where the protein can exist in a dilute solution-like environment. In summary, microgels that interact weakly with proteins do not strongly influence protein dynamics or stability because these large microgels constitute an upper size limit on crowding effects.

Full text

RESEARC H ARTIC L E Open Access
An upper limit for macromolecular crowding
effects
Andrew C Miklos
1
, Conggang Li
1,2
, Courtney D Sorrell
3,4,5
, L Andrew Lyon
3,4
and Gary J Pielak
1,6,7*
Abstract
Background: Solutions containing high macromolecule concentrations are predicted to affect a number of protein
properties compared to those properties in dilute solution. In cells, these macromolecular crowders have a large
range of sizes and can occupy 30% or more of the available volume. We chose to study the stability and ps-ns
internal dynamics of a globular protein whose radius is ~2 nm when crowded by a synthetic microgel composed
of poly(N-isopropylacrylamide-co-acrylic acid) with particle radii of ~300 nm.
Results: Our studies revealed no change in protein rotational or ps-ns backbone dynamics and only mild
(~0.5 kcal/mol at 37°C, pH 5.4) stabilization at a volume occupancy of 70%, which approaches the occupancy of
closely packing spheres. The lack of change in rotational dynamics indicates the absence of strong crowder-protein
interactions.
Conclusions: Our observations are explained by the large size discrepancy between the protein and crowders and
by the internal structure of the microgels, which provide interstitial spaces and internal pores where the protein
can exist in a dilute solution-like environment. In summary, microgels that interact weakly with proteins do not
strongly influence protein dynamics or stability because these large microgels constitute an upper size limit on
crowding effects.
Background
The cellular interior, where most biological processes
occur, is unlike the dilute solutions where most proteins
are studied. The large volume excluded by high macro-
molecule concentrations in cells, from 20-40% [1], is
predicted to change many protein properties compared
to dilute solution. We used a synthetic microgel com-
posed of poly(N-isopropylacrylamide-co-acrylic acid)
[p-NIPAm-co-AAc (Figure 1A)], as a crowding agent to
study the backbone dynamics and the stability of the
globular test protein, chymotrypsin inhibitor 2 (CI2).
p-NIPAm-co-AAc is of interest in pharmaceutical
applications because it forms environmentally sensitive
microgels [2]. Each microgel particle (Figure 1B) is a
lightly cross-linked single polymer molecule of molecu-
lar weight 10
9
Da with an average of 70 monomer units
between each cross link. The polymer absorbs a large
amount of water resulting in spherical particles of
300 nm radii that exclude large amounts of solution
volume. Their porosity arises from the balance between
the external (solution) osmotic pressure and the internal
osmotic pressure. This internal pressure is the result of
the solvated cations that neutralize the deprotonated
polymer side chains. We chose this crowding agent
because its status as a drug delivery molecule makes it
pharmaceutically relevant, and its ability to take up
water provides a model for volume exclusion by a mole-
cule much larger than our test protein.
CI2 is a small globular protein (7.4 kDa, PDB ID:
2CI2) that exhibits two-state folding [3]. NMR relaxa-
tion experiments [4] allowed us to assess backbone rota-
tional dynamics for CI2 in the presence and absence of
p-NIPAm-co-AAc. Amide proton exchange experiments
[5,6] allowed us to assess the stability of CI2 in dilute
and crowded conditions.
Globular proteins are often treated like hard spheres,
buttheyhavemeasurableamounts of internal motion.
Analysis of relaxation parameters from NMR experi-
ments - longitudinal and transverse relaxation times, T
1
and T
2
,andthe
15
N-
1
H nuclear Overhauser effect
* Correspondence: gary_pielak@unc.edu
1
Department of Chemistry, University of North Carolina, Chapel Hill, North
Carolina 27599, USA
Full list of author information is available at the end of the article
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© 2011 Miklos et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creative commons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, pro vided the original work is properly cited.

(NOE) of backbone
15
N atoms - offers a residue-level
window into this ps-ns backbone motion. The analysis
involves a model-free method established by Lipari and
Szabo [7]. Analysis is performed by fitting the spectral
density function I(ω) as calculated from measured T
1
,
T
2
, and NOE values [8], to the equation [7]
I(ω)= 2
5S2τm
1+ω2τ2
m
+(1 −S2)τ
1+ω2τ2

The overall correlation time τis linked to the correla-
tion time for isotropic tumbling, τ
m
, and internal motion
timescale, τ
e
, by the equation
1
τ=1
τ
e
+1
τ
m
with the internal motions faster than the overall iso-
tropic tumbling. The order parameter, S
2
,canhave
values between 0 and 1, and is related to the degree of
internal mobility for a particular
1
H-
15
Nvector.AnS
2
value of 0 corresponds to complete freedom of motion.
In this instance, relaxation is related solely to internal
motion. An S
2
value of 1 corresponds to complete
restriction of the vector with respect to overall molecule
motion, and relaxation is related solely to isotropic tum-
bling of the protein. These parameters can be linked to
models for motion, in our case, the “wobble-in-a-cone”
model [7]. Variations of Lipari-Szabo analysis exist for
cases involving ms timescale conformational exchange,
but no CI2 residue (except Thr40) has significant contri-
butions from slow exchange [9]. It is also possible to
study the equilibrium thermodynamic stability of globu-
lar proteins by using NMR.
Amide proton exchange experiments can be used
with NMR to assess protein stability. The technique
relies on the exchange of amide protons for deuterons
in a D
2
O solution. We have recently reviewed the
requirements for its application in crowded solution by
using NMR [10].
Exchange occurs via the scheme
cl −1H
k
op
−→
←−
k
cl
op −1Hkint
−→ op −2H(Scheme 1
)
with opening rate k
op
,closingratek
cl
,andrateof
exchange from the open state k
int
. If the protein is stable
(k
cl
>>k
op
) and exchange from the open state is rate
limiting, the stability of an amide proton against
exchange (G0∗
o
p
) can be determined with the equation,
G0∗
op =−RT ln kobs
k
int
where Ris the gas constant and Tis the absolute tem-
perature. The value of k
obs
, the overall rate of exchange
for any particular backbone amide proton, is assessed by
acquiring
1
H-
15
N heteronuclear single quantum correla-
tion (HSQC) spectra as a function of time after initiat-
ing exchange. As with dynamics, G0∗
o
p
can be quantified
on a per-residue basis. The largest G0
∗
o
p
values match
the global protein stability values determined by other
methods (e.g., calorimetry, circular dichroism spectropo-
larimetry) [11].
Results
Experiments were performed by using samples compris-
ing 1 mM CI2 in 50 mM sodium acetate solution, pH
5.4 at 37°C. Crowded samples also contained 10 g/L
p-NIPAm-co-AAc microgels.
Polymer Characterization
The microgels composed of p-NIPAm-co-AAc have an
average hydrodynamic radius (R
H
) of 312 nm and an
average polydispersity of 7.4%. The molecular weight of
the microgels was estimated to be 1 GDa by multiple
angle laser light scattering [12].
Figure 1 Structure and Size of p-NIPAm-co-AAc:A)The
monomeric repeat of NIPAm. B) Overall shape and size of p-NIPAm-
co-AAc microgels.
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Controls for Amide Proton Exchange
To determine whether exchange from the open state
(k
int
) is rate limiting, nuclear Overhauser enchancement
spectroscopy-detected amide proton exchange (NOESY-
HEX) experiments were performed [10]. The results are
given in Table 1, along with individual backbone residue
decay rates from HSQC-detected amide proton
exchange.
To determine whether k
int
values are changed by
crowding, phase-modulated clean exchange (CLEANEX-
PM) experiments [13] were used to determine k
int
for
residues on the extended loop region of CI2. For His37,
k
int
values were 11 ± 2 s
-1
in dilute solution and 8 ± 2 s
-1
in 10 g/L p-NIPAm-co-AAc.
Dynamics
Analysis of the T
1
,T
2
, and NOE data (Additional File 1)
acquired in dilute solution and in 10 g/L p-NIPAm-co-
AAc yielded the values for τ
m
,S
2
,andτ
e
.Thevalueofτ
m
was the same (4.1 ns) in dilute solution and in 10 g/L
p-NIPAm-co-AAc, and is consistent with the value
obtained by Shaw et al. in dilute solution [9]. Histograms
of S
2
and τ
e
versus residue number are shown in Figure 2.
Linear least squares analysis of a plot of S
2
in dilute solu-
tion versus S
2
in crowded solution gives a slope of 1.0 ±
0.1, a y-intercept of 0.1 ± 0.1 and an R
2
value of 0.80.
Amide Proton Exchange and Stability
Values for k
obs
were determined in triplicate for solutions
in the presence and absence of 10 g/L p-NIPAm-co-AAc.
Exchange was slowed in 10 g/L p-NIPAm-co-AAc com-
pared to dilute solution (Figure 3). Values of ΔG
0*op
were
determined by using values for k
int
calculated from
SPHERE [14] and k
obs
values from amide proton
exchange experiments. A listing of values is given in
Additional File 2. A histogram of ΔG
0*op
versus residue
number is shown in Figure 4.
Discussion
Thevolumeoccupancyofp-NIPAm-co-AAc solutions
defines the degree of crowding. Using a hydrodynamic
radius of 312 nm and a molecular weight of 1 GDa, the
microgel in a 10 g/L solution occupies ~70% of the solution
volume at pH 5.4 and 37°C (the conditions used in our
experiments). The practical limit of spherical packing is
64% volume occupancy [15], but soft materials such as
microgels can be “overpacked”[16]. Our solutions, however,
were still in the liquid state, meaning our value for volume
occupancy is likely an overestimate. The high value does,
however, suggest that experimental conditions were within
the realm of crowding, as other systems show crowding
effects at less than 20% volume occupancy [17,18].
Although the microgel slowed exchange (Figure 3), it
was necessary to perform control experiments to ensure
Table 1 NOESY-HEX results
Residue(s) k
obs
NOESY (s
-1
×10
5
)k
obs
HSQC (s
-1
×10
5
)
Leu8 3 3
Val9 2 2
Leu8 + Val9
a
55
Leu8, Val9
b
5 N/A
Lys17 52 40
Lys18 20 15
Lys17 + Lys18
a
72 55
Lys17, Lys18
b
50 N/A
Ala58 3 4
Glu59 3 3
Ala58 + Glu59
a
67
Ala58, Glu59
b
7 N/A
k
abs
values from NOESY-detected amide proton exchange and HSQC-detected
amide proton exchange for CI2 in 10 g/L p-NIPAm-co-AAc, 50 mM sodium
acetate, pH 5.4, 37°C.
a
Sum of values from individual crosspeak decays.
b
Exchange rate of amide-amide NOESY crosspeak.
Figure 2 CI2 dynamics in p-NIPAm-co-AAc: Order parameters
(upper panel) and timescales of internal motion (lower panel) for
CI2 in dilute solution and in 10 g/L NIPAm-AAc.
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that stability values could be obtained under both sets of
conditions. First, we confirmed that amide proton
exchange from the open state (k
int
, Scheme 1) is rate
limiting. Under this condition, pairs of proximal amide
protons, A and B, open with the same frequency, but
with different k
int
values. That is, amide proton
exchanges for A and B are uncorrelated. By observing
the decay of an amide-amide NOESY crosspeak corre-
sponding to a resonance coupling between A and B, it is
possible to determine whether their exchange is corre-
lated or uncorrelated. If the exchange is uncorrelated,
the decay curve of the amide-amide crosspeak should
equal the product of the individual amide proton decay
curves [19,20],
I
AB
=I
A
·I
B
In this instance, the overall exchange rate of the
amide-amide crosspeak will correspond to the sum of
the individual exchange rate constants,
K
AB
=K
A
+K
B
All these rates can be assessed from a series of
15
N-fil-
tered
1
H-
1
H NOESY spectra acquired under exchange
conditions [10].
As shown in Table 1, the exchange rates observed for
the amide-amide crosspeaks for CI2 in both dilute solu-
tion and in 10 g/L p-NIPAm-co-AAc are, within the
uncertainty of the experiment, the sums of their respec-
tive individual exchange rates, indicating that the
exchanges are uncorrelated. We conclude that exchange
from the open state is rate limiting, allowing determina-
tion of stability from amide proton exchange rates.
Second, we must determine if the microgel changes
k
int
from the values determined in dilute solution. The
dilute solution value for each residue is calculated by
using the computer program, SPHERE [14] (http://www.
fccc.edu/research/labs/roder/sphere/). The program uses
values from the exchange of free peptides [21], and
relies solely on the primary structure of the test protein.
We assessed whether k
int
is affected by adding
p-NIPAm-co-AAc by using the CLEANEX-PM experi-
ment [13]. We measured the exchange rate of the His37
amide proton, which is fully exposed in the flexible loop
region of CI2 (residues 35-44). The data indicated that
the intrinsic rate of exchange in 10 g/L p-NIPAm-co-
AAc (8 ± 2 s
-1
) is within uncertainty of the value in
dilute solution (11 ± 2 s
-1
). These results suggest that
k
int
values can be used without alteration. Having shown
that it is valid to use k
obs
and k
int
values to obtain open-
ing free energies, we constructed histograms of ΔG
0*op
values versus residue number (Figure 4).
Dynamics and Stability
Crowding involves two different types of effects on pro-
tein stability: volume exclusion and chemical interactions.
Volume exclusion is expected to stabilize protein native
states, whereas chemical interactions can be stabilizing or
destabilizing [6]. Attractive chemical interactions are
expected to impede rotational dynamics, and the micro-
gel used here is known to have favorable electrostatic
interactions with proteins at low ionic strength [22]. Our
data were collected at pH 5.4, where the microgel is
negatively charged. The truncated form of CI2 we use
has an isoelectric point (pI) of 6. Therefore, the polymer
Figure 3 Exchange Curves: Amide proton exchange curves for
Lys24 in dilute solution (blue triangles) and in 10 g/L p-NIPAm-co-
AAc (green squares). Values for k
obs
are 7.53 ± 0.05 × 10
-5
±s
-1
in
dilute solution and 4.55 ± 0.05 × 10
-5
s
-1
in 10 g/L p-NIPAm-co-AAc.
These uncertainties are from non-linear least squares fitting and are
smaller than the uncertainty from triplicate analysis.
Figure 4 CI2 stability in p-NIPAm-co-AAc: Results are shown for
dilute solution (blue) and 10 g/L p-NIPAm-co-AAc (green). Error bars
reflect the standard error in k
obs
values from three trials. Colored
arrows indicate the average ΔG
0*op
values for globally exchanging
residues [20] in crowded (5.2 kcal/mol) and dilute (4.9 kcal/mol)
solution.
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and CI2 are oppositely charged, and one might expect an
attractive interaction.
Our observation that the order parameters (S
2
), the
timescale of internal motion (τ
e
), and the rotational cor-
relation time (τ
m
), are unchanged by the polymer indi-
cates the absence of significant chemical interactions
between the polymer and CI2. The lack of interaction
probably arises because we used an ionic strength of 50
mM, which minimizes binding [22]. Therefore, we only
consider contributions from volume exclusion effects.
The patterns of ΔG
0*op
values along the amino acid
sequence (Figure 4) are the same in dilute solution as
they are in the microgel solution, suggesting that the
microgel does not alter the open states of CI2. The
ΔG
0*op
values in the microgel are uniformly larger than
the values for dilute solution, indicating the polymer sta-
bilizes the protein with a maximal stability increase of
approximately 0.4 kcal/mol. Averaging the ΔG
0*op
values
from residues known to be implicated in global unfold-
ing [20] show that the microgel increases the overall sta-
bility from 4.9 kcal/mol to 5.2 kcal/mol. We cannot
state with certainty that the increased stability arises
from the polymeric nature of the microgel because its
crosslinked nature makes determination of a suitable
monomer unit difficult.
Considering the volume fraction estimate of ~70%, a 0.3
kcal/mol stability increase is quite small. A modest
increase is anticipated, however, because the hydrodynamic
radius of CI2 is only 1% that of the p-NIPAm-co-AAc
microgels (Figure 5). In such a system, CI2 can occupy
interstitial spaces between p-NIPAm-co-AAc microgels,
putting CI2 in a dilute solution environment. Alternatively,
the microgel particles probably have pores large enough to
accommodate CI2 and water.
Next, we try to relate the stability change to the back-
bone dynamics data (Figure 2). The data indicate that
the increased stability does not alter the ps-ns backbone
dynamics. It has been proposed that stability changes
are associated with alterations of ps-ns backbone
dynamics [23,24]. Our results do not indicate a connec-
tion, because we observe increased stability without a
change in ps-ns timescale dynamics. The most straight-
forward conclusion is that stability is not linked to back-
bone ps-ns dynamics. It is possible, however, that
stability is reflected in slower (ms-s) motions [25].
Conclusions
Even though the 10 g/L solution of p-NIPAm-co-AAc
microgels occupy ~70% of solution volume, these condi-
tions do not affect the ps-ns timescale backbone
dynamics of CI2. The microgel, however, does have a
modest stabilizing effect on the protein. These conclu-
sions are explained by the fact that the majority of the
protein occupies a water-like environment in interstitial
spaces of the microgel particles. In the context of
p-NIPAm-co-AAc as a drug delivery tool, this is promising
information, supporting the notion that these microgels
are biocompatible materials. It seems likely, however, that
larger crowding agents such as p-NIPAm-co-AAc can
have more noticeable effects when present in mixed solu-
tions that also contain multiple sizes of crowders [26].
Methods
15
N-enriched CI2 was expressed and purified as
described by Miklos et al. [10].
Polymer Synthesis and Characterization
A general synthesis for NIPAm-AAc microgels is
described by Jones and Lyon [27], but variations yield
products with different properties (size, temperature, pH
dependence, etc.) [28-30]. The microgels used here were
prepared via aqueous, surfactant-free, free radical precipi-
tation polymerization using 70 mM total monomer con-
centration. Briefly, N-isopropylacrylamide (0.6973 g) and
N,N’-methylenebis(acrylamide) (0.0215 g) were dissolved
in 99 mL of H2O and filtered through a 0.8 μmsyringe
filter into a round bottom flask. The mixture was
bubbled with N
2
(g) and heated to 70°C (± 2°C) over
~1 h. Acrylic acid (46 μL) was then added. Polymeriza-
tion was initiated by adding a solution of (NH
4
)
2
S
2
O
8
(0.0226 g) dissolved in 1 mL of H
2
O. This reaction was
stirredat70°C(±2°C)underablanketofN
2
(g) for 4 h
Figure 5 Interstitial Spaces in p-NIPAm-co-AAc: Depiction of the
scale of microgel sizes for p-NIPAm-co-AAc (green) and CI2 (red).
CI2 can exist in the spaces between crowder particles or within
pores (of unknown size) without experiencing a change in
environment compared to bulk water.
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and was stirred and cooled overnight. The mixture was
filtered through Whatman #2 paper and stored. Ali-
quots of the resultant colloidal dispersion were purified
with centrifugation at 15,000 × g, decanted, and resus-
pended in H
2
O. This process was performed three
times. The particles were then lyophilized to yield a
white powder.
The microgels were characterized after suspension in
sodium acetate (pH 5.4) and passage through a 0.8 μm
filter. This solution was sonicated for 5 min, allowed to
equilibrate for 30 min, then analyzed by using multi-
angle laser light scattering (MALLS) [12].
NMR
HSQC-detected and NOESY-HEX experiments were
performed on a 500 MHz Varian Inova spectrometer
equipped with a triple-resonance HCN cold probe as
described by Miklos et al. [10]. CLEANEX-PM experi-
ments were conducted as described by Hwang et al.
[13] with a 600 MHz Varian Inova spectrometer
equipped with a triple-resonance HCN probe with
three-axis gradients system.
15
NT
1
and T
2
relaxation times and
15
N{
1
H} NOEs
were measured as described by Kay et al. [31]. Experi-
ments were performed on the 600 MHz spectrometer.
Lipari-Szabo model free analysis [7] was performed with
the software package Relaxn 2.2. [32]. The majority of
residues were fit with the original model-free formalism
[4] to yield τ
m
,S
2
and τ
e
.
Additional material
Additional file 1:
15
NT
1
,
15
NT
2
, and
1
H-
15
N NOEs for CI2. A table
containing
15
NT
1
,
15
NT
2
, and
1
H-
15
N NOE values for CI2 in dilute
solution and 10 g/L p-NIPAm-co-AAc at 37°C, pH 5.4.
Additional file 2: CI2 Stability Values. A table containing ΔG
0*op
values
and standard error from triplicate results for CI2 in 10 g/L p-NIPAm-co-
AAc at 37°C, pH 5.4.
Acknowledgements
This work was supported by the National Institutes of Health (5DP1OD783)
and the National Science Foundation (MCB-1051819). We thank Gregory B.
Young for spectrometer assistance and Elizabeth Pielak for helpful
comments on the manuscript.
Author details
1
Department of Chemistry, University of North Carolina, Chapel Hill, North
Carolina 27599, USA.
2
State Key Laboratory of Magnetic Resonance and
Molecular and Atomic Physics, Wuhan Institute of Physics and Mathematics,
Chinese Academy of Sciences, Wuhan, 430071, PR China.
3
School of
Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta, GA
30332, USA.
4
Petit Institute for Bioengineering and Bioscience, Georgia
Institute of Technology, Atlanta, GA 30332, USA.
5
Department of Chemistry,
University of Alberta, Edmonton, AB, T6G 2G2, Canada.
6
Department of
Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North
Carolina 27599, USA.
7
Lineberger Comprehensive Cancer Center, University
of North Carolina, Chapel Hill, North Carolina 27599, USA.
Authors’contributions
ACM, CL, LAL, and GJP designed the research; ACM and CL performed the
NMR experiments; CDS prepared and characterized microgels; ACM and GJP
wrote the manuscript; All authors read and approved the final manuscript.
Received: 15 December 2010 Accepted: 31 May 2011
Published: 31 May 2011
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doi:10.1186/2046-1682-4-13
Cite this article as: Miklos et al.: An upper limit for macromolecular
crowding effects. BMC Biophysics 2011 4:13.
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