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1.  The Effect of Macromolecular Crowding on the Electrostatic Component of Barnase–Barstar Binding: A Computational, Implicit Solvent-Based Study 
PLoS ONE  2014;9(6):e98618.
Macromolecular crowding within the cell can impact both protein folding and binding. Earlier models of cellular crowding focused on the excluded volume, entropic effect of crowding agents, which generally favors compact protein states. Recently, other effects of crowding have been explored, including enthalpically-related crowder–protein interactions and changes in solvation properties. In this work, we explore the effects of macromolecular crowding on the electrostatic desolvation and solvent-screened interaction components of protein–protein binding. Our simple model enables us to focus exclusively on the electrostatic effects of water depletion on protein binding due to crowding, providing us with the ability to systematically analyze and quantify these potentially intuitive effects. We use the barnase–barstar complex as a model system and randomly placed, uncharged spheres within implicit solvent to model crowding in an aqueous environment. On average, we find that the desolvation free energy penalties incurred by partners upon binding are lowered in a crowded environment and solvent-screened interactions are amplified. At a constant crowder density (fraction of total available volume occupied by crowders), this effect generally increases as the radius of model crowders decreases, but the strength and nature of this trend can depend on the water probe radius used to generate the molecular surface in the continuum model. In general, there is huge variation in desolvation penalties as a function of the random crowder positions. Results with explicit model crowders can be qualitatively similar to those using a lowered “effective” solvent dielectric to account for crowding, although the “best” effective dielectric constant will likely depend on multiple system properties. Taken together, this work systematically demonstrates, quantifies, and analyzes qualitative intuition-based insights into the effects of water depletion due to crowding on the electrostatic component of protein binding, and it provides an initial framework for future analyses.
PMCID: PMC4051634  PMID: 24915485
2.  Effects of Macromolecular Crowding on Protein Conformational Changes 
PLoS Computational Biology  2010;6(7):e1000833.
Many protein functions can be directly linked to conformational changes. Inside cells, the equilibria and transition rates between different conformations may be affected by macromolecular crowding. We have recently developed a new approach for modeling crowding effects, which enables an atomistic representation of “test” proteins. Here this approach is applied to study how crowding affects the equilibria and transition rates between open and closed conformations of seven proteins: yeast protein disulfide isomerase (yPDI), adenylate kinase (AdK), orotidine phosphate decarboxylase (ODCase), Trp repressor (TrpR), hemoglobin, DNA β-glucosyltransferase, and Ap4A hydrolase. For each protein, molecular dynamics simulations of the open and closed states are separately run. Representative open and closed conformations are then used to calculate the crowding-induced changes in chemical potential for the two states. The difference in chemical-potential change between the two states finally predicts the effects of crowding on the population ratio of the two states. Crowding is found to reduce the open population to various extents. In the presence of crowders with a 15 Å radius and occupying 35% of volume, the open-to-closed population ratios of yPDI, AdK, ODCase and TrpR are reduced by 79%, 78%, 62% and 55%, respectively. The reductions for the remaining three proteins are 20–44%. As expected, the four proteins experiencing the stronger crowding effects are those with larger conformational changes between open and closed states (e.g., as measured by the change in radius of gyration). Larger proteins also tend to experience stronger crowding effects than smaller ones [e.g., comparing yPDI (480 residues) and TrpR (98 residues)]. The potentials of mean force along the open-closed reaction coordinate of apo and ligand-bound ODCase are altered by crowding, suggesting that transition rates are also affected. These quantitative results and qualitative trends will serve as valuable guides for expected crowding effects on protein conformation changes inside cells.
Author Summary
The biophysical properties of proteins inside cells can be expected to be quite different from those typically measured by in vitro experiments in dilute solutions. In particular, intracellular macromolecular crowding may significantly affect the equilibria and transition rates between different conformations of a protein, and hence its functions. What are the trends and magnitudes of such crowding effects? We address this question here by applying a recently developed approach for modeling crowding. Seven proteins, each with structures for both an open state and a closed state, are studied. Crowding exerts significant effects on the open-closed equilibria of four proteins and more modest effects on the remaining three. Potentials of mean force along the open-closed reaction coordinate, and hence transition rates, are similarly affected. The extent of conformational changes is the main determinant for the magnitudes of crowding effects, but the protein size also plays an important role. The effects of crowding become stronger as the protein size increases. Conformational transitions of the ribosome, an extremely large complex, during translation are predicted to experience particularly strong effects of intracellular crowding. We conclude that deduction of intracellular behaviors from in vitro experiments requires explicit consideration of crowding effects.
PMCID: PMC2895631  PMID: 20617196
3.  Macromolecular Crowding as a Suppressor of Human IAPP Fibril Formation and Cytotoxicity 
PLoS ONE  2013;8(7):e69652.
The biological cell is known to exhibit a highly crowded milieu, which significantly influences protein aggregation and association processes. As several cell degenerative diseases are related to the self-association and fibrillation of amyloidogenic peptides, understanding of the impact of macromolecular crowding on these processes is of high biomedical importance. It is further of particular relevance as most in vitro studies on amyloid aggregation have been performed in diluted solution which does not reflect the complexity of their cellular surrounding. The study presented here focuses on the self-association of the type-2 diabetes mellitus related human islet amyloid polypeptide (hIAPP) in various crowded environments including network-forming macromolecular crowding reagents and protein crowders. It was possible to identify two competing processes: a crowder concentration and type dependent stabilization of globular off-pathway species and a – consequently - retarded or even inhibited hIAPP fibrillation reaction. The cause of these crowding effects was revealed to be mainly excluded volume in the polymeric crowders, whereas non-specific interactions seem to be most dominant in protein crowded environments. Specific hIAPP cytotoxicity assays on pancreatic β-cells reveal non-toxicity for the stabilized globular species, in contrast to the high cytotoxicity imposed by the normal fibrillation pathway. From these findings it can be concluded that cellular crowding is able to effectively stabilize the monomeric conformation of hIAPP, hence enabling the conduction of its normal physiological function and prevent this highly amyloidogenic peptide from cytotoxic aggregation and fibrillation.
PMCID: PMC3726762  PMID: 23922768
4.  An FFT-based method for modeling protein folding and binding under crowding: benchmarking on ellipsoidal and all-atom crowders 
Journal of chemical theory and computation  2013;9(10):10.1021/ct4005195.
It is now well recognized that macromolecular crowding can exert significant effects on protein folding and binding stability. In order to calculate such effects in direct simulations of proteins mixed with bystander macromolecules, the latter (referred to as crowders) are usually modeled as spheres and the proteins represented at a coarse-grained level. Our recently developed postprocessing approach allows the proteins to be represented at the all-atom level but, for computational efficiency, has only been implemented for spherical crowders. Modeling crowder molecules in cellular environments and in vitro experiments as spheres may distort their effects on protein stability. Here we present a new method that is capable for treating aspherical crowders. The idea, borrowed from protein-protein docking, is to calculate the excess chemical potential of the proteins in crowded solution by fast Fourier transform (FFT). As the first application, we studied the effects of ellipsoidal crowders on the folding and binding free energies of all-atom proteins, and found, in agreement with previous direct simulations with coarse-grained protein models, that the aspherical crowders exert greater stabilization effects than spherical crowders of the same volume. Moreover, as demonstrated here, the FFT-based method has the important property that its computational cost does not increase strongly even when the level of details in representing the crowders is increased all the way to all-atom, thus significantly accelerating realistic modeling of protein folding and binding in cell-like environments.
PMCID: PMC3811151  PMID: 24187527
5.  Volume Exclusion and Soft Interaction Effects on Protein Stability under Crowded Conditions† 
Biochemistry  2010;49(33):6984-6991.
Most proteins function in nature under crowded conditions, and crowding can change protein properties. Quantification of crowding effects, however, is difficult because solutions containing hundreds of grams per liter of macromolecules often interfere with observing the protein being studied. Models for macromolecular crowding tend to focus on the steric effects of crowders, neglecting potential chemical interactions between the crowder and the test protein. Here, we report the first systematic, quantitative, residue-level study of crowding effects on the equilibrium stability of a globular protein. We used a system comprising poly(vinylpyrrolidone)s (PVPs) of varying molecular weights as crowding agents and chymotrypsin inhibitor 2 (CI2) as a small globular test protein. Stability was quantified with NMR-detected amide 1H exchange. We analyze the data in terms of hard particle exclusion, confinement, and soft interactions. For all crowded conditions, nearly every observed residue experiences a stabilizing effect. The exceptions are residues where stabilities are unchanged. At a PVP concentration of 100 g/L, the data are consistent with theories of hard particle exclusion. At higher concentrations, the data are more consistent with confinement. The data show that the crowder also stabilizes the test protein by weakly binding its native state. We conclude that the role of native-state binding and other soft interactions need to be seriously considered when applying both theory and experiment to studies of macromolecular crowding.
PMCID: PMC2927838  PMID: 20672856
6.  Macromolecular crowding remodels the energy landscape of a protein by favoring a more compact unfolded state 
Journal of the American Chemical Society  2010;132(30):10445-10452.
The interior of cells is highly crowded with macromolecules, which impacts all physiological processes. To explore how macromolecular crowding may influence cellular protein folding, we interrogated the folding landscape of a model β-rich protein, cellular retinoic acid-binding protein I (CRABP I), in the presence of an inert crowding agent (Ficoll 70). Urea titrations revealed a crowding-induced change in the water-accessible polar amide surface of its denatured state, based on an observed ca. 15% decrease in the m-value (the change in unfolding free energy with respect to urea concentration), and the effect of crowding on the equilibrium stability of CRABP I was less than our experimental error (i.e., ≤1.2 kcal/mol). Consequently, we directly probed the effect of crowding on the denatured state of CRABP I by measuring side chain accessibility using iodide quenching of tryptophan fluorescence and chemical modification of cysteines. We observed that the urea-denatured state is more compact under crowded conditions, and the observed extent of reduction of the m value by crowding agent is fully consistent with the extent of reduction of the accessibility of the Trp and Cys probes, suggesting a random and nonspecific compaction of the unfolded state. The thermodynamic consequences of crowding-induced compaction are discussed. In addition, over a wide range of Ficoll concentration, crowding significantly retarded the unfolding kinetics of CRABP I without influencing the urea dependence of the unfolding rate, arguing for no appreciable change in the nature of the transition state. Our results demonstrate how macromolecular crowding may influence protein folding by effects both on the unfolded state ensemble and on unfolding kinetics. (end of abstract)
PMCID: PMC2921862  PMID: 20662522
7.  Conformational Sampling of Peptides in the Presence of Protein Crowders from AA/CG-Multiscale Simulations 
The Journal of Physical Chemistry. B  2012;116(29):8610-8620.
Macromolecular crowding is recognized as an important factor influencing folding and conformational dynamics of proteins and nucleic acids. Previous views of crowding have focused on the mostly entropic volume exclusion effect of crowding but recent studies are indicating the importance of enthalpic effects, in particular changes in electrostatic interactions due to crowding. Here, temperature replica exchange molecular dynamics simulations of trp-cage and melittin in the presence of explicit protein crowders are presented to further examine the effect of protein crowders on peptide dynamics. The simulations involve a three-component multiscale modeling scheme where the peptides are represented at atomistic level, the crowder proteins at a coarse-grained level, and the surrounding aqueous solvent as implicit solvent. This scheme optimally balances a physically realistic description for the peptide with computational efficiency. The multiscale simulations were compared with simulations of the same peptides in different dielectric environments with dielectric constants ranging from 5 to 80. It is found that the sampling in the presence of the crowders resembles sampling with reduced dielectric constants between 10 and 40. Furthermore, diverse conformational ensembles are generated in the presence of crowders including partially unfolded states for trp-cage. These findings emphasize the importance of enthalpic interactions over volume exclusion effects in describing the effects of cellular crowding.
PMCID: PMC3398202  PMID: 22429139
protein-protein interactions; molecular dynamics; coarse-graining; multiscale simulations; replica exchange; crowding
8.  Effects of Macromolecular Crowding on the Conformational Ensembles of Disordered Proteins 
The journal of physical chemistry letters  2013;4(20):10.1021/jz401817x.
Due to their conformational malleability, intrinsically disordered proteins (IDPs) are particularly susceptible to influences of crowded cellular environments. Here we report a computational study of the effects of macromolecular crowding on the conformational ensemble of a coarse-grained IDP model, by using two approaches. In one, the IDP is simulated along with the crowders; in the other, crowder-free simulations are postprocessed to predict the conformational ensembles under crowding. We found significant decreases in the radius of gyration of the IDP under crowding, and suggest repulsive interactions with crowders as a common cause for chain compaction in a number of experimental studies. The postprocessing approach accurately reproduced the conformational ensembles of the IDP in the direct simulations here, and holds enormous potential for realistic modeling of IDPs under crowding, by permitting thorough conformation sampling for the proteins even when they and the crowders are both represented at the all-atom level.
PMCID: PMC3846091  PMID: 24312701
Intrinsically disordered proteins; macromolecular crowding; postprocessing approach
9.  Nonaddtive Effects of Mixed Crowding on Protein Stability 
Proteins  2009;77(1):133-138.
The crowded environments inside cells can have significant effects on the folding stability and other biophysical properties of proteins. In this study on how macromolecular crowding affects protein folding, we took a significant step toward realistically mimicking intracellular environments by using a mixture of two crowding agents, Ficoll and dextran. We found that the mixed crowding exerts a greater stabilizing effect than the sum of the two individual crowding agents. Therefore, the composition of crowders, not just the total concentration, has a significant influence on the effects of crowding on protein folding. Since the composition of intracellular macromolecules varies within the lifetime of a cell, our finding may provide an explanation for age being an important risk factor for protein aggregation-related diseases such as Alzheimer’s disease and Parkinson’s disease.
PMCID: PMC4456354  PMID: 19408299
Protein stability; protein folding; macromolecular crowding; mixed crowding; FKBP
10.  A Didactic Model of Macromolecular Crowding Effects on Protein Folding 
PLoS ONE  2010;5(8):e11936.
A didactic model is presented to illustrate how the effect of macromolecular crowding on protein folding and association is modeled using current analytical theory and discrete molecular dynamics. While analytical treatments of crowding may consider the effect as a potential of average force acting to compress a polypeptide chain into a compact state, the use of simulations enables the presence of crowding reagents to be treated explicitly. Using an analytically solvable toy model for protein folding, an approximate statistical thermodynamic method is directly compared to simulation in order to gauge the effectiveness of current analytical crowding descriptions. Both methodologies are in quantitative agreement under most conditions, indication that both current theory and simulation methods are capable of recapitulating aspects of protein folding even by utilizing a simplistic protein model.
PMCID: PMC2914742  PMID: 20689808
11.  Reduced native state stability in crowded cellular environment due to protein-protein interactions 
The effect of cellular crowding environments on protein structure and stability is a key issue in molecular and cellular biology. The classical view of crowding emphasizes the volume exclusion effect that generally favors compact, native states. Here, results from molecular dynamics simulations and NMR experiments show that protein crowders may destabilize native states via protein-protein interactions. In the model system considered here, mixtures of villin head piece and protein G at high concentrations, villin structures become increasingly destabilized upon increasing crowder concentrations. The denatured states observed in the simulation involve partial unfolding as well as more subtle conformational shifts. The unfolded states remain overall compact and only partially overlap with unfolded ensembles at high temperature and in the presence of urea. NMR measurements on the same systems confirm structural changes upon crowding based on changes of chemical shifts relative to dilute conditions. An analysis of protein-protein interactions and energetic aspects suggests the importance of enthalpic and solvation contributions to the crowding free energies that challenge an entropic-centered view of crowding effects.
PMCID: PMC3601481  PMID: 23402619
Molecular dynamics simulation; folding; villin; protein-protein interactions
12.  A method for computing association rate constants of atomistically represented proteins under macromolecular crowding 
Physical biology  2012;9(6):066008.
In cellular environments, two protein molecules on their way to form a specific complex encounter many bystander macromolecules. The latter molecules, or crowders, affect both the energetics of the interaction between the test molecules and the dynamics of their relative motion. In earlier work (Zhou and Szabo 1991 J. Chem. Phys. 95 5948-52), it has been shown that, in modeling the association kinetics of the test molecules, the presence of crowders can be accounted for by their energetic and dynamic effects. The recent development of the transient-complex theory for protein association in dilute solutions makes it possible to easily incorporate the energetic and dynamic effects of crowders. The transient complex refers to a late on-pathway intermediate, in which the two protein molecules have near-native relative separation and orientation but have yet to form the many short-range specific interactions of the native complex. The transient-complex theory predicts the association rate constant as ka = ka0exp (−ΔGel*/kBT), where ka0 is the “basal” rate constant for reaching the transient complex by unbiased diffusion, and the Boltzmann factors captures the influence of long-range electrostatic interactions between the protein molecules. Crowders slow down the diffusion, therefore reducing the basal rate constant (to kac0), and induce an effective interaction energy ΔGc. We show that the latter interaction energy for atomistic proteins in the presence of spherical crowders is “long”-ranged, allowing the association rate constant under crowding to be computed as kac = kac0exp[−(ΔGel* + ΔGc*)/kBT]. Applications demonstrate that this computational method allows for realistic modeling of protein association kinetics under crowding.
PMCID: PMC3521150  PMID: 23197255
association rate constant; transient complex; macromolecular crowding; diffusion; electrostatic rate enhancement
13.  Reconstituting Intracellular Vesicle Fusion Reactions: The Essential Role of Macromolecular Crowding 
Journal of the American Chemical Society  2015;137(40):12873-12883.
Intracellular vesicle fusion is mediated by SNAREs and Sec1/Munc18 (SM) proteins. Despite intensive efforts, the SNARE-SM mediated vesicle fusion reaction has not been faithfully reconstituted in biochemical assays. Here, we present an unexpected discovery that macromolecular crowding is required for reconstituting the vesicle fusion reaction in vitro. Macromolecular crowding is known to profoundly influence the kinetic and thermodynamic behaviors of macromolecules, but its role in membrane transport processes such as vesicle fusion remains unexplored. We introduced macromolecular crowding agents into reconstituted fusion reactions to mimic the crowded cellular environment. In this crowded assay, SNAREs and SM proteins acted in concert to drive efficient membrane fusion. In uncrowded assays, by contrast, SM proteins failed to associate with the SNAREs and the fusion rate decreased more than 30-fold, close to undetectable levels. The activities of SM proteins were strictly specific to their cognate SNARE isoforms and sensitive to biologically relevant mutations, further supporting that the crowded fusion assay accurately recapitulates the vesicle fusion reaction. Using this crowded fusion assay, we also showed that the SNARE-SM mediated fusion reaction can be modulated by two additional factors: NSF and α-SNAP. These findings suggest that the vesicle fusion machinery likely has been evolutionarily selected to function optimally in the crowded milieu of the cell. Accordingly, macromolecular crowding should constitute an integral element of any reconstituted fusion assay.
Graphical abstract
PMCID: PMC4638180  PMID: 26431309
14.  Effects of Molecular Crowding on the Dynamics of Intrinsically Disordered Proteins 
PLoS ONE  2012;7(11):e49876.
Inside cells, the concentration of macromolecules can reach up to 400 g/L. In such crowded environments, proteins are expected to behave differently than in vitro. It has been shown that the stability and the folding rate of a globular protein can be altered by the excluded volume effect produced by a high density of macromolecules. However, macromolecular crowding effects on intrinsically disordered proteins (IDPs) are less explored. These proteins can be extremely dynamic and potentially sample a wide ensemble of conformations under non-denaturing conditions. The dynamic properties of IDPs are intimately related to the timescale of conformational exchange within the ensemble, which govern target recognition and how these proteins function. In this work, we investigated the macromolecular crowding effects on the dynamics of several IDPs by measuring the NMR spin relaxation parameters of three disordered proteins (ProTα, TC1, and α-synuclein) with different extents of residual structures. To aid the interpretation of experimental results, we also performed an MD simulation of ProTα. Based on the MD analysis, a simple model to correlate the observed changes in relaxation rates to the alteration in protein motions under crowding conditions was proposed. Our results show that 1) IDPs remain at least partially disordered despite the presence of high concentration of other macromolecules, 2) the crowded environment has differential effects on the conformational propensity of distinct regions of an IDP, which may lead to selective stabilization of certain target-binding motifs, and 3) the segmental motions of IDPs on the nanosecond timescale are retained under crowded conditions. These findings strongly suggest that IDPs function as dynamic structural ensembles in cellular environments.
PMCID: PMC3506533  PMID: 23189168
15.  Molecular Crowding Favors Reactivity of a Human Ribozyme Under Physiological Ionic Conditions 
Biochemistry  2013;52(46):10.1021/bi400816s.
In an effort to relate RNA folding to function under cellular-like conditions, we monitored the self-cleavage reaction of the human hepatitis delta virus (HDV)-like CPEB3 ribozyme in the background of physiological ionic concentrations and various crowding and cosolute agents. We found that under physiological free Mg2+ concentrations (~0.1 to 0.5 mM Mg2+), both crowders and cosolutes stimulate the rate of self-cleavage, up to ~6-fold, but that in 10 mM Mg2+—conditions widely used for in vitro ribozyme studies—these same additives have virtually no effect on self-cleavage rate. We further observe a dependence of self-cleavage rate on crowder size, wherein rate stimulation is diminished for crowders larger than the size of the unfolded RNA. Monitoring effects of crowding and cosolute agents on rates in biological amounts of urea revealed additive-promoted increases in both low and high Mg2+ concentrations, with a maximal stimulation of more than 10-fold and a rescue of the rate to its urea-free values. Small-angle X-ray scattering (SAXS) experiments reveal a structural basis for this stimulation in that higher molecular weight crowding agents favor a more compact form of the ribozyme in 0.5 mM Mg2+ that is essentially equivalent to the form under standard ribozyme conditions of 10 mM Mg2+ and no crowder. This finding suggests that at least a portion of the rate enhancement arises from favoring the native RNA tertiary structure. We conclude that cellular-like crowding supports ribozyme reactivity by favoring a compact form of the ribozyme, but only under physiological ionic and cosolute conditions.
PMCID: PMC3882164  PMID: 24187989
16.  Modulation of Calmodulin Plasticity by the Effect of Macromolecular Crowding 
Journal of molecular biology  2009;391(5):933-943.
In vitro biochemical reactions are most often studied in dilute solution, a poor mimic of the intracellular space of eukaryotic cells which are crowded with mobile and immobile macromolecules. Such crowded conditions exert volume exclusion and other entropic forces that have the potential to impact chemical equilibria and reaction rates. In this article, we used the well characterized and ubiquitous molecule calmodulin (CaM) and a combination of theoretical and experimental approaches to address how crowding impacts CaM’s conformational plasticity. CaM is a dumbbell shaped molecule that contains four EF hands (two in the N-lobe and two in the C-lobe) that each could bind Ca2+ leading to stabilization of certain substates that favor interactions with other target proteins. Using coarse-grained molecular simulations, we explored the distribution of CaM conformations in the presence of crowding agents. These predictions in which crowding effects enhances the population of compact structures were then confirmed in experimental measurements using fluorescence resonance energy transfer techniques of donor/acceptor labeled CaM under normal and crowded conditions. We further explored the folding energy landscape and examined the structural characteristics of CaM at free energy basins using protein reconstruction methods. We discovered that crowding stabilizes several different compact conformations, which reflects the inherent plasticity in CaM’s structure. From these results, we suggest that the EF hands in the C-lobe are flexible and can be thought of as a switch, while those in the N-lobe are stiff as analogous to a rheostat. New combinatorial signaling properties may arise from the product of the differential plasticity of the two distinct lobes of CaM in the presence of crowding. We discuss the implications of these results for modulating CaM’s ability to bind Ca2+ and target proteins.
PMCID: PMC2728162  PMID: 19577574
Calmodulin; EF-hands; macromolecular crowding; coarse-grained molecular simulations; FRET; Ca2+-signaling
17.  Simulation and Modeling of Crowding Effects on the Thermodynamic and Kinetic Properties of Proteins with Atomic Details 
Biophysical reviews  2013;5(2):207-215.
Recent experimental studies of protein folding and binding under crowded solutions suggest that crowding agents exert subtle influences on the thermodynamic and kinetic properties of the proteins. While some of the crowding effects can be understood qualitatively from simple models of the proteins, quantitative rationalization of these effects requires an atomistic representation of the protein molecules in modeling their interactions with crowders. A computational approach, known as postprocessing, has opened the door for atomistic modeling of crowding effects. This review summarizes the applications of the postprocessing approach for studying crowding effects on the thermodynamics and kinetics of protein folding, conformational transition, and binding. The integration of atomistic modeling with experiments in crowded solutions promises new insight into biochemical processes in cellular environments.
PMCID: PMC3659821  PMID: 23710260
macromolecular crowding; protein folding; protein binding; simulation; modeling; postprocessing
18.  Protein Folding Requires Crowd Control in a Simulated Cell 
Journal of Molecular Biology  2010;397(5):1329-1338.
Macromolecular crowding has a profound effect upon biochemical processes in the cell. We have computationally studied the effect of crowding upon protein folding for 12 small domains in a simulated cell using a coarse-grained protein model, which is based upon Langevin dynamics, designed to unify the often disjoint goals of protein folding simulation and structure prediction. The model can make predictions of native conformation with accuracy comparable with that of the best current template-free models. It is fast enough to enable a more extensive analysis of crowding than previously attempted, studying several proteins at many crowding levels and further random repetitions designed to more closely approximate the ensemble of conformations. We found that when crowding approaches 40% excluded volume, the maximum level found in the cell, proteins fold to fewer native-like states. Notably, when crowding is increased beyond this level, there is a sudden failure of protein folding: proteins fix upon a structure more quickly and become trapped in extended conformations. These results suggest that the ability of small protein domains to fold without the help of chaperones may be an important factor in limiting the degree of macromolecular crowding in the cell. Here, we discuss the possible implications regarding the relationship between protein expression level, protein size, chaperone activity and aggregation.
PMCID: PMC2891488  PMID: 20149797
TM, template modelling; macromolecular crowding; protein structure prediction; protein misfolding; protein aggregation; protein expression
19.  Cell-Free Protein Expression under Macromolecular Crowding Conditions 
PLoS ONE  2011;6(12):e28707.
Cell-free protein expression (CFPE) comprised of in vitro transcription and translation is currently manipulated in relatively dilute solutions, in which the macromolecular crowding effects present in living cells are largely ignored. This may not only affect the efficiency of protein synthesis in vitro, but also limit our understanding of the functions and interactions of biomolecules involved in this fundamental biological process.
Methodology/Principal Findings
Using cell-free synthesis of Renilla luciferase in wheat germ extract as a model system, we investigated the CFPE under macromolecular crowding environments emulated with three different crowding agents: PEG-8000, Ficoll-70 and Ficoll-400, which vary in chemical properties and molecular size. We found that transcription was substantially enhanced in the macromolecular crowding solutions; up to 4-fold increase in the mRNA production was detected in the presence of 20% (w/v) of Ficoll-70. In contrast, translation was generally inhibited by the addition of each of the three crowding agents. This might be due to PEG-induced protein precipitation and non-specific binding of translation factors to Ficoll molecules. We further explored a two-stage CFPE in which transcription and translation was carried out under high then low macromolecular crowding conditions, respectively. It produced 2.2-fold higher protein yield than the coupled CFPE control. The macromolecular crowding effects on CFPE were subsequently confirmed by cell-free synthesis of an approximately two-fold larger protein, Firefly luciferase, under macromolecular crowding environments.
Three macromolecular crowding agents used in this research had opposite effects on transcription and translation. The results of this study should aid researchers in their choice of macromolecular crowding agents and shows that two-stage CFPE is more efficient than coupled CFPE.
PMCID: PMC3234285  PMID: 22174874
20.  An upper limit for macromolecular crowding effects 
BMC Biophysics  2011;4:13.
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.
PMCID: PMC3120801  PMID: 21627822
21.  Influence of Nanoparticle Size and Shape on Oligomer Formation of an Amyloidogenic Peptide 
Understanding the influence of macromolecular crowding and nanoparticles on the formation of in-register β-sheets, the primary structural component of amyloid fibrils, is a first step towards describing in vivo protein aggregation and interactions between synthetic materials and proteins. Using all atom molecular simulations in implicit solvent we illustrate the effects of nanoparticle size, shape, and volume fraction on oligomer formation of an amyloidogenic peptide from the transthyretin protein. Surprisingly, we find that inert spherical crowding particles destabilize in-register β-sheets formed by dimers while stabilizing β-sheets comprised of trimers and tetramers. As the radius of the nanoparticle increases crowding effects decrease, implying smaller crowding particles have the largest influence on the earliest amyloid species. We explain these results using a theory based on the depletion effect. Finally, we show that spherocylindrical crowders destabilize the ordered β-sheet dimer to a greater extent than spherical crowders, which underscores the influence of nanoparticle shape on protein aggregation.
PMCID: PMC3117580  PMID: 21691423
crowding; in vivo; early events; amyloid
22.  Effects of Macromolecular Crowding on Amyloid Beta (16–22) Aggregation Using Coarse-Grained Simulations 
The Journal of Physical Chemistry. B  2014;118(47):13513-13526.
To examine the effect of crowding on protein aggregation, discontinuous molecular dynamics (DMD) simulations combined with an intermediate resolution protein model, PRIME20, were applied to a peptide/crowder system. The systems contained 192 Aβ(16–22) peptides and crowders of diameters 5, 20, and 40 Å, represented here by simple hard spheres, at crowder volume fractions of 0.00, 0.10, and 0.20. Results show that both crowder volume fraction and crowder diameter have a large impact on fibril and oligomer formation. The addition of crowders to a system of peptides increases the rate of oligomer formation, shifting from a slow ordered formation of oligomers in the absence of crowders, similar to nucleated polymerization, to a fast collapse of peptides and subsequent rearrangement characteristic of nucleated conformational conversion with a high maximum in the number of peptides in oligomers as the total crowder surface area increases. The rate of conversion from oligomers to fibrils also increases with increasing total crowder surface area, giving rise to an increased rate of fibril growth. In all cases, larger volume fractions and smaller crowders provide the greatest aggregation enhancement effects. We also show that the size of the crowders influences the formation of specific oligomer sizes. In our simulations, the 40 Å crowders enhance the number of dimers relative to the numbers of trimers, hexamers, pentamers, and hexamers, while the 5 Å crowders enhance the number of hexamers relative to the numbers of dimers, trimers, tetramers, and pentamers. These results are in qualitative agreement with previous experimental and theoretical work.
PMCID: PMC4254002  PMID: 25347801
23.  Structure of Metaphase Chromosomes: A Role for Effects of Macromolecular Crowding 
PLoS ONE  2012;7(4):e36045.
In metaphase chromosomes, chromatin is compacted to a concentration of several hundred mg/ml by mechanisms which remain elusive. Effects mediated by the ionic environment are considered most frequently because mono- and di-valent cations cause polynucleosome chains to form compact ∼30-nm diameter fibres in vitro, but this conformation is not detected in chromosomes in situ. A further unconsidered factor is predicted to influence the compaction of chromosomes, namely the forces which arise from crowding by macromolecules in the surrounding cytoplasm whose measured concentration is 100–200 mg/ml. To mimic these conditions, chromosomes were released from mitotic CHO cells in solutions containing an inert volume-occupying macromolecule (8 kDa polyethylene glycol, 10.5 kDa dextran, or 70 kDa Ficoll) in 100 µM K-Hepes buffer, with contaminating cations at only low micromolar concentrations. Optical and electron microscopy showed that these chromosomes conserved their characteristic structure and compaction, and their volume varied inversely with the concentration of a crowding macromolecule. They showed a canonical nucleosomal structure and contained the characteristic proteins topoisomerase IIα and the condensin subunit SMC2. These observations, together with evidence that the cytoplasm is crowded in vivo, suggest that macromolecular crowding effects should be considered a significant and perhaps major factor in compacting chromosomes. This model may explain why ∼30-nm fibres characteristic of cation-mediated compaction are not seen in chromosomes in situ. Considering that crowding by cytoplasmic macromolecules maintains the compaction of bacterial chromosomes and has been proposed to form the liquid crystalline chromosomes of dinoflagellates, a crowded environment may be an essential characteristic of all genomes.
PMCID: PMC3335069  PMID: 22540018
24.  Computer Simulations of the Bacterial Cytoplasm 
Biophysical reviews  2013;5(2):109-119.
Ever since the pioneering work of Minton, it has been recognized that the highly crowded interior of biological cells has the potential to cause dramatic changes to both the kinetics and thermodynamics of protein folding and association events relative to behavior that might be observed in dilute solution conditions. One very productive way to explore the effects of crowding on protein behavior has been to use macromolecular crowding agents that exclude volume without otherwise strongly interacting with the protein under study. An alternative, complementary approach to understanding the potential differences between behavior in vivo and in vitro is to develop simulation models that explicitly attempt to model intracellular environments at the molecular scale, and that thereby can be used to directly monitor biophysical behavior in conditions that accurately mimic those encountered in vivo. It is with studies of this type that the present review will be concerned. We review in detail four published studies that have attempted to simulate the structure and dynamics of the bacterial cytoplasm and that have each explored different biophysical aspects of the cellular interior. While each of these studies has yielded important new insights, there are important questions that remain to be resolved in terms of determining the relative contributions made by energetic and hydrodynamic interactions to the diffusive behavior of macromolecules and to the thermodynamics of protein folding and associations in vivo. Some possible new directions for future generation simulation models of the cytoplasm are outlined.
PMCID: PMC3728174  PMID: 23914257
computer simulation; bacterial cytoplasm; macromolecular crowding; energetic interactions; hydrodynamic interactions
25.  Interactions of Macromolecular Crowding Agents and Cosolutes with Small-Molecule Substrates: Effect on Horseradish Peroxidase Activity with Two Different Substrates 
The Journal of Physical Chemistry. B  2014;118(36):10624-10632.
The importance of solution composition on enzymatic reactions is increasingly appreciated, particularly with respect to macromolecular cosolutes. Macromolecular crowding and its effect on enzymatic reactions has been studied for several enzymes and is often understood in terms of changes to enzyme conformation. Comparatively little attention has been paid to the chemical properties of small-molecule substrates for enzyme reactions in crowded solution. In this article, we studied the reaction of horseradish peroxidase (HRP) with two small-molecule substrates that differ in their hydrophobicity. Crowding agents and cosolutes had quite different effects on HRP activity when the substrate used was 3,3′,5,5′-tetramethylbenzidine (TMB, which is hydrophobic) as compared to o-phenylenediamine (OPD, which is more hydrophilic). Reaction rates with TMB were much more sensitive to the presence of crowding agents and cosolutes than OPD, suggesting that the small-molecule substrates may themselves be interacting with crowders and cosolutes. At high polyethylene glycol (PEG) concentrations (25–30 wt/wt %), no reaction was observed for TMB. Even at lower concentrations, Michaelis constants (KM) for HRP with the more hydrophobic substrate increased in the presence of crowding agents and cosolutes, particularly with PEG. Diffusion of TMB and OPD in the PEG and dextran reaction media was evaluated using pulsed field gradient nuclear magnetic resonance (PFG-NMR). The diffusivity of the TMB decreased 3.9× in 10% PEG 8k compared to that in buffer and decreased only 1.7× for OPD. Together, these data suggest that weak attractive interactions between small-molecule substrates and crowders or cosolutes can reduce substrate chemical activity and consequently decrease enzyme activity and that these effects vary with the identity of the molecules involved. Because many enzymes can act on multiple substrates, it is important to consider substrate chemistry in understanding enzymatic reactions in complex media such as biological fluids.
PMCID: PMC4161143  PMID: 25157999

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