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.
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.
protein-protein interactions; molecular dynamics; coarse-graining; multiscale simulations; replica exchange; crowding
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.
Macromolecular crowding inside cells affects the thermodynamic and kinetic properties of proteins. The scaled particle theory (SPT) has played an important role toward establishing a qualitative picture for the effects of crowding. However, SPT-based modeling lacks molecular details. Molecular dynamics simulations overcome this limitation, but at great computational cost. Here we present a theoretical method for modeling crowding at the atomic level. The method makes it possible to achieve exhaustive conformational sampling in modeling crowding effects and to tackle challenges posed by large protein oligomers and by complex mixtures of crowders.
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.
crowding; in vivo; early events; amyloid
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.
How the crowded environment inside cells affects folding, stability and structures of proteins is a vital question, since most proteins are made and function inside cells. Here we describe how crowded conditions can be created in vitro and in silico and how we have used this to probe effects on protein properties. We have found that folded forms of proteins become more compact in the presence of macromolecular crowding agents; if the protein is aspherical, the shape also changes (extent dictated by native-state stability and chemical conditions). It was also discovered that the shape of the macromolecular crowding agent modulates the folding mechanism of a protein; in addition, the extent of asphericity of the protein itself is an important factor in defining its folding speed.
Macromolecular crowding; Ficoll® 70; energy landscape theory; off-lattice model; excluded volume effect; protein folding mechanism; spectroscopy
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.
TM, template modelling; macromolecular crowding; protein structure prediction; protein misfolding; protein aggregation; protein expression
In cells proteins fold and unfold in the presence of macromolecules with various sizes and shapes. Recent experiments by Liang and co-workers (J Biol Chem 2004;279:55109–55116; J Mol Biol 2006; 364:469–482) show that protein refolding is enhanced by a mixture of two different crowding agents relative to the individual crowding agents, and an optimal mixing ratio exists. Here we present a theory that predicts the existence of an optimal mixing ratio. The theory is based on models for calculating the changes in the chemical potentials of the folded and unfolded states by a mixture of crowders. The existence of an optimal mixing ratio results from the dependences of these chemical-potential changes on crowder sizes and concentrations, which can be argued to be quite general. We further predict that, for any crowding agent, the stabilizing effect can be optimized both by varying the molecular weight and by varying the mixing ratio of two species with different molecular weights.
excluded-volume effect; folding stability; folding rate
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)
Cytoplasm contains a large number of macromolecules at extremely high densities. How this striking nature of intracellular milieu called macromolecular crowding affects intracellular signaling remains uncharacterized. Here, we examined the effect of macromolecular crowding on ERK MAPK phosphorylation by MEK MAPKK. Addition of polyethylene glycol-6000 (PEG-6000) as a crowder to mimic intracellular environments, elicited a biphasic response to the overall ERK phosphorylation rate. Furthermore, probability of processive phosphorylation (processivity) of tyrosine and threonine residues within the activation loop on ERK increased non-linearly for increasing PEG-6000 concentration. Based on the experimental data, we developed for the first time a mathematical model integrating all of the effects of thermodynamic activity, viscosity, and processivity in crowded media, and found that ERK phosphorylation is transition-state-limited reaction. The mathematical model allows accurate estimation of the effects of macromolecular crowding on a wide range of reaction kinetics, from transition-state-limited to diffusion-limited reactions.
The internal dynamics of proteins inside of cells may be
affected by the crowded intracellular environments. Here, we test
a novel approach to simulations of crowding, in which simulations
in the absence of crowders are postprocessed to predict crowding effects,
against the direct approach of simulations in the presence of crowders.
The effects of crowding on the flap dynamics of HIV-1 protease predicted
by the postprocessing approach are found to agree well with those
calculated by the direct approach. The postprocessing approach presents
distinct advantages over the direct approach in terms of accuracy
and speed and is expected to have broad impact on atomistic simulations
of macromolecular crowding.
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.
Confinement and crowding are two major factors that can potentially impact protein folding in cellular environments. Theories based on considerations of excluded volumes predict disparate effects on protein folding stability for confinement and crowding: confinement can stabilize proteins by over 10kBT but crowding has a very modest effect on stability. On the other hand, confinement and crowding are both predicted to favor conformations of the unfolded state which are compact, and consequently may increase the folding rate. These predictions are largely borne out by experimental studies of protein folding under confined and crowded conditions in the test tube. Protein folding in cellular environments is further complicated by interactions with surrounding surfaces and other factors. Concerted theoretical modeling and test-tube and in vivo experiments promise to elucidate the complexity of protein folding in cellular environments.
Proteins perform their function in cells where macromolecular solutes reach concentrations of >300 g/L and occupy >30% of the volume. The volume excluded by these macromolecules will stabilize globular proteins because the native state occupies less space than the denatured state. Theory predicts that crowding can increase the ratio of folded to unfolded protein by a factor of 100, amounting to 3 kcal/mol of stabilization at room temperature. We tested the idea that volume exclusion dominates the crowding effect in cells with a variant of protein L, a 7-kDa globular protein with seven lysine residues replaced by glutamic acids. Eighty-four percent of the variant molecules populate the denatured state in dilute buffer at room temperature, compared to 0.1% for the wild-type protein. We then used in-cell nuclear magnetic resonance spectroscopy to show that the cytoplasm of Escherichia coli does not overcome even this modest (~1 kcal/mol) free energy deficit. The data are consistent with the idea that non-specific interactions between cytoplasmic components can overcome the excluded volume effect. Evidence for these interactions is provided by the observation that adding simple salts folds the variant in dilute solution, but increasing the salt concentration inside E. coli does not fold the protein. Our data are consistent with other studies of protein stability in cells, and suggest that stabilizing excluded volume effects, which must be present under crowded conditions, can be ameliorated by non-specific interactions between cytoplasmic components.
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.
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.
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.
The interior of cells is crowded thus making it important to assess the effects of macromolecules on the folding of proteins. Using the Self-Organized Polymer (SOP) model, which is a coarse-grained representation of polypeptide chains, we probe the mechanical stability of Ubiquitin (Ub) monomers and trimers ((Ub)3) in the presence of monodisperse spherical crowding agents. Crowding increases the volume fraction (Φc)-dependent average force (⟨fu(Φc)⟩), relative to the value at Φc = 0, needed to unfold Ub and the polyprotein. For a given Φc, the values of ⟨fu(Φc)⟩ increase as the diameter (σc) of the crowding particles decreases. The average unfolding force ⟨fu(Φc)⟩ depends on the ratio DRg, where D≈σc(π6Φc)13 with Rg being the radius of gyration of Ub (or (Ub)3) in the unfolded state. Examination of the unfolding pathways shows that, relative to Φc = 0, crowding promotes reassociation of ruptured secondary structural elements. Both the nature of the unfolding pathways and ⟨fu(Φc)⟩ for (Ub)3 are altered in the presence of crowding particles with the effect being most dramatic for the subunit that unfolds last. We predict, based on SOP simulations and theoretical arguments, that 〈fu(Φc)〉~Φc13ν, where ν is the Flory exponent that describes the unfolded (random coil) state of the protein.
Depletion Effect; Entropic Stabilization; Crowders; AFM; Loading Rate; SOP model
Microenvironments of biological cells are dominated in vivo by macromolecular crowding and resultant excluded volume effects. This feature is absent in dilute in vitro cell culture. Here, we induced macromolecular crowding in vitro by using synthetic macromolecular globules of nm-scale radius at physiological levels of fractional volume occupancy. We quantified the impact of induced crowding on the extracellular and intracellular protein organization of human mesenchymal stem cells (MSCs) via immunocytochemistry, atomic force microscopy (AFM), and AFM-enabled nanoindentation. Macromolecular crowding in extracellular culture media directly induced supramolecular assembly and alignment of extracellular matrix proteins deposited by cells, which in turn increased alignment of the intracellular actin cytoskeleton. The resulting cell-matrix reciprocity further affected adhesion, proliferation, and migration behavior of MSCs. Macromolecular crowding can thus aid the design of more physiologically relevant in vitro studies and devices for MSCs and other cells, by increasing the fidelity between materials synthesized by cells in vivo and in vitro.
Macromolecular crowding is one of the key characteristics of the cellular environment and therefore, is intimately coupled to the process of protein folding in vivo. While previous studies have provided invaluable insight into the effect of crowding on the stability and folding rate of protein tertiary structures, very little is known about how crowding affects protein folding dynamics at the secondary structure level. Herein, we examine the thermal stability and folding-unfolding kinetics of three small folding motifs, i.e., a 34-residue α-helix, a 34-residue cross-linked helix-turn-helix, and a 16-residue β hairpin, in the presence of two commonly used crowding agents, Dextran 70 (200 g/L) and Ficoll 70 (200 g/L). We find that these polymers do not induce any appreciable changes in the folding kinetics of the two helical peptides, which is somewhat surprising as the helix-coil transition kinetics have been shown to depend on viscosity. Also to our surprise and in contrast to what has been observed for larger proteins, we find that crowding leads to an appreciable decrease in the folding rate of the shortest β-hairpin peptide, indicating that besides the excluded volume effect, other factors also need to be considered when evaluating the net effect of crowding on protein folding kinetics. A model considering both the static and dynamic effects arising from the presence of the crowding agent is proposed to rationalize these results.
Macromolecular crowding; confinement; protein folding; helix-coil transition; β-hairpin folding
Expected and observed effects of volume exclusion on the free energy of rigid and flexible macromolecules in crowded and confined systems, and consequent effects of crowding and confinement on macromolecular reaction rates and equilibria are summarized. Findings from relevant theoretical/simulation and experimental literature published from 2004 onward are reviewed. Additional complexity arising from the heterogeneity of local environments in biological media, and the presence of nonspecific interactions between macromolecules over and above steric repulsion are discussed. Theoretical and experimental approaches to the characterization of crowding- and confinement-induced effects in systems approaching the complexity of living organisms are suggested.
excluded volume; configurational entropy; free energy; protein-protein interactions; macromolecular associations; protein folding; site-binding
The intracellular environment imposes a variety of constraints on biochemical reaction systems that can substantially change reaction rates and equilibria relative to an ideal solution-based environment. One of the most notable features of the intracellular environment is its dense macromolecular crowding, which, among many other effects, tends to strongly enhance binding and assembly reactions. Despite extensive study of biochemistry in crowded media, it remains extremely difficult to predict how crowding will quantitatively affect any given reaction system due to the dependence of the crowding effect on numerous assumptions about the reactants and crowding agents involved. We previously developed a two dimensional stochastic off-lattice model of binding reactions based on the Green’s function reaction dynamics (GFRD) method in order to create a versatile simulation environment in which one can explore interactions among many parameters of a crowded assembly system. In the present work, we examine interactions among several critical parameters for a model dimerization system: the total concentration of reactants and inert particles, the binding probability upon a collision between two reactant monomers, the mean time of dissociation reactions, and the diffusion coefficient of the system. Applying regression models to equilibrium constants across parameter ranges shows that the effect of the total concentration is approximately captured by a low-order nonlinear polynomial model, while the other three parameter effects are each accurately captured by a linear model. Furthermore, validation on tests with multi-parameter variations reveals that the effects of these parameters are separable from one another over a broad range of variation in all four parameters. The simulation work suggests that predictive models of crowding effects can accommodate a wider variety of parameter variations than prior theoretical models have so far achieved.
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.
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.
Enzymatic activities in vivo occur in a crowded environment composed of
many macromolecules. This environment influences DNA replication by increasing the concentration of
the constituents, desolvation, decreasing the degrees of freedom for diffusion and hopping of
proteins onto DNA, and enhancing binding equilibria and catalysis. However, the effect of
macromolecular crowding on protein structure is poorly understood. Here we examine macromolecular
crowding using the replication system of bacteriophage T7 and we show that it affects several
aspects of DNA replication; the activity of DNA helicase increases and the sensitivity of DNA
polymerase to salt is reduced. We also demonstrate, using SAXS analysis, that the complex between
DNA helicase and DNA polymerase/trx is far more compact in a crowded environment. The highest
enzymatic activity corresponds to the most compact structure. Better knowledge of the effect of
crowding on structure and activity will enhance mechanistic insight beyond information obtained from
NMR and X-ray structures.
Macromolecular crowding; T7 replisome; DNA polymerase; Helicase; SAXS
The cytosol of a cell is a concentrated milieu of a variety of different molecules, including small molecules (salts and metabolites) and macromolecules such as nucleic acids, polysaccharides, proteins and large macromolecular complexes. Macromolecular crowding in the cytosolic environment is proposed to influence various properties of proteins, including substrate binding affinity and enzymatic activity. Here we chose to use the synthetic crowding agent Ficoll, which is commonly used to mimic cytosolic crowding conditions to study the crowding effect on the catalytic properties of glycolytic enzymes, namely phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, and acylphosphatase. We determined the kinetic parameters of these enzymes in the absence and in the presence of the crowding agent. We found that the Michaelis constant, Km, and the catalytic turnover number, kcat, of these enzymes are not perturbed by the presence of the crowding agent Ficoll. Our results support earlier findings which suggested that the Michaelis constant of certain enzymes evolved in consonance with the substrate concentration in the cell to allow effective enzyme function in bidirectional pathways. This conclusion is further supported by the analysis of nine other enzymes for which the Km values in the presence and absence of crowding agents have been measured.