"Macromolecular crowding": thermodynamic consequences for protein-protein interactions within the T4 DNA replication complex

In vitro biochemical assays are typically performed using very dilute solutions of macromolecular components. On the other hand, total intracellular concentrations of macromolecular solutes are very high, resulting in an in vivo environment that is significantly "volume-occupied." In vitro studies with the DNA replication proteins of bacteriophage T4 have revealed anomalously weak binding of T4 gene 45 protein to the rest of the replication complex. We have used inert macromolecular solutes to mimic typical intracellular solution conditions of high volume occupancy to investigate the effects of "macromolecular crowding" on the binding equilibria involved in the assembly of the T4 polymerase accessory proteins complex. The same approach was also used to study the assembly of this complex with T4 DNA polymerase (gene 43 protein) and T4 single-stranded DNA binding protein (gene 32 protein) to form the five protein "holoenzyme". We find that the apparent association constant (Ka) of gene 45 for gene 44/62 proteins in forming both the accessory protein complex and the holoenzyme increases markedly (from approximately 7 x 10(6) to approximately 3.5 x 10(8) M-1) as a consequence of adding polymers such as polyethylene glycol and dextran. Although the processivity of the polymerase alone is not directly effected by the addition of such polymers to the solution, macromolecular crowding does significantly stabilize the holoenzyme and thus indirectly increases the observed processivity of the holoenzyme complex. The use of macromolecular crowding to increase the stability of multienzyme complexes in general is discussed, as is the relevance of these results to DNA replication in vivo.     

Macromolecular crowding in biological systems: hydrodynamics and NMR methods

Most biologically relevant environments involve highly concentrated macromolecular solutions and most biological processes involve macromolecules that diffuse and interact with other macromolecules. Macromolecular crowding is a general phenomenon that strongly affects the transport properties of macromolecules (rotational and translational diffusion) as well as the position of their equilibria. NMR methods can provide information on molecular interactions, as well as on translational and rotational diffusion. In fact, rotational diffusion, through its determinant role in NMR relaxation, places a practical limit on the systems that can be studied by NMR. While in dilute solutions of non-aggregating macromolecules this limit is set by macromolecular size, in crowded solutions excluded volume effects can have a strong effect on the observed diffusion rates. Hydrodynamic theory offers some insight into the magnitude of crowding effects on NMR observable parameters.     

Metabolic buffering exerted by macromolecular crowding on DNA-DNA interactions: origin and physiological significance

Crowding, which characterizes the interior of all living cells, has been shown to dramatically affect biochemical processes, leading to stabilization of compact morphologies, enhanced macromolecular associations, and altered reaction rates. Due to the crowding-mediated shift in binding equilibria toward association, crowding agents were proposed to act as a metabolic buffer, significantly extending the range of intracellular conditions under which interactions occur. Crowding may, however, impose a liability because, by greatly and generally enhancing macromolecular association, it can lead to irreversible interactions. To better understand the physical determinants and physiological consequences of crowding-mediated buffering, we studied the effects of crowding, or excluded volume, on DNA structures. Results obtained from isothermal titration calorimetry (ITC) and UV melting experiments indicate that crowding-induced effects are marginal under conditions that a priori favor association of DNA strands but become progressively larger when conditions deteriorate. As such, crowding exerts "genuine" buffering activity. Unexpectedly, crowding-mediated effects are found to include enthalpy terms that favorably contribute to association processes. We propose that these enthalpy terms and preferential stabilization derive from a reconfiguration of DNA hydration that occurs in dense DNA-rich phases obtained in crowded environments.     

Dynamics of unfolded polypeptide chains in crowded environment studied by fluorescence correlation spectroscopy

Proteins have evolved to fold and function within a cellular environment that is characterized by high macromolecular content. The earliest step of protein folding represents intrachain contact formation of amino acid residues within an unfolded polypeptide chain. It has been proposed that macromolecular crowding can have significant effects on rates and equilibria of biomolecular processes. However, the kinetic consequences on intrachain diffusion of polypeptides have not been tested experimentally, yet. Here, we demonstrate that selective fluorescence quenching of the oxazine fluorophore MR121 by the amino acid tryptophan (Trp) in combination with fast fluorescence correlation spectroscopy (FCS) can be used to monitor end-to-end contact formation rates of unfolded polypeptide chains. MR121 and Trp were incorporated at the terminal ends of polypeptides consisting of repetitive units of glycine (G) and serine (S) residues. End-to-end contact formation and dissociation result in "off" and "on" switching of MR121 fluorescence and underlying kinetics can be revealed in FCS experiments with nanosecond time resolution. We revisit previous experimental studies concerning the dependence of end-to-end contact formation rates on polypeptide chain length, showing that kinetics can be described by Gaussian chain theory. We further investigate effects of solvent viscosity and temperature on contact formation rates demonstrating that intrachain diffusion represents a purely diffusive, entropy-controlled process. Finally, we study the influence of macromolecular crowding on polypeptide chain dynamics. The data presented demonstrate that intrachain diffusion is fast in spite of hindered diffusion caused by repulsive interactions with macromolecules. Findings can be explained by effects of excluded volume reducing chain entropy and therefore accelerating the loop search process. Our results suggest that within a cellular environment the early formation of structural elements in unfolded proteins can still proceed quite efficiently in spite of hindered diffusion caused by high macromolecular content.     

Connecting the Dots: The Effects of Macromolecular Crowding on Cell Physiology

The physicochemical properties of cellular environments with a high macromolecular content have been systematically characterized to explain differences observed in the diffusion coefficients, kinetics parameters, and thermodynamic properties of proteins inside and outside of cells. However, much less attention has been given to the effects of macromolecular crowding on cell physiology. Here, we review recent findings that shed some light on the role of crowding in various cellular processes, such as reduction of biochemical activities, structural reorganization of the cytoplasm, cytoplasm fluidity, and cellular dormancy. We conclude by presenting some unresolved problems that require the attention of biophysicists, biochemists, and cell physiologists. Although it is still underappreciated, macromolecular crowding plays a critical role in life as we know it.     

Noise-reduction through interaction in gene expression and biochemical reaction processes

We demonstrate that interaction in gene expression and biochemical reaction processes has a significant influence on reducing fluctuations. Especially, we have found that the interaction between synthesized proteins and background molecules can reduce the fluctuation level in gene expression, which is a counter example to the intuition that background factors disturb information processing in genetic networks by increasing the noise level. This fact also indicates that the macromolecular crowding observed in actual cells can contribute to reduce the noise level. In addition, the noise-reduction phenomenon is not limited to the interaction between the proteins and background molecules, but can be applied to other reactions such as a dimerization process and the coupling of reactions with large fluctuations by intrinsic noise. Finally, on the basis of these results, we propose a new and plausible method for reducing the fluctuations generated in synthesized genetic networks, and also discuss the applicability of this method to the stabilization of system dynamics by using a toggle switch model.     

Protein folding and binding in confined spaces and in crowded solutions

Simple theoretical models are presented to illustrate the effects of spatial confinement and macromolecular crowding on the equilibria and rates of protein folding and binding. Confinement is expected to significantly stabilize the folded state, but for crowding only a marginal effect on protein stability is expected. In confinement the unfolded chain is restricted to a cage but in crowding the unfolded chain may explore different interstitial voids. Because confinement and crowding eliminate the more expanded conformations of the unfolded state, folding from the compact unfolded state is expected to speed up. Crowding will shift the binding equilibrium of proteins toward the bound state. The significant slowing down in protein diffusion by crowding, perhaps beneficial for chaperonin action, could result in a decrease in protein binding rates.     

DNA condensation in bacteria: Interplay between macromolecular crowding and nucleoid proteins

The volume of a typical Eschericia coli nucleoid is roughly 10⁴ times smaller than the volume of a freely coiling linear DNA molecule with the same length as the E. coli genome. We review the main forces that have been suggested to contribute to this compaction factor: macromolecular crowding (that “pushes” the DNA together), DNA charge neutralization by various polycationic species (that “glues” the DNA together), and finally, DNA deformations due to DNA supercoiling and nucleoid proteins. The direct contributions of DNA supercoiling and nucleoid proteins to the total compaction factor are probably small. Instead, we argue that the formation of the bacterial nucleoid can be described as a consequence of the influence of macromolecular crowding on thick, supercoiled protein-DNA fibers, that have been partly charge neutralized by small multivalent cations.     

The effects of polydisperse crowders on the compaction of the Escherichia coli nucleoid

DNA binding proteins, supercoiling, macromolecular crowders, and transient DNA attachments to the cell membrane have all been implicated in the organization of the bacterial chromosome. However, it is unclear what role these factors play in compacting the bacterial DNA into a distinct organelle-like entity, the nucleoid. By analyzing the effects of osmotic shock and mechanical squeezing on Escherichia coli, we show that macromolecular crowders play a dominant role in the compaction of the DNA into the nucleoid. We find that a 30% increase in the crowder concentration from physiological levels leads to a three-fold decrease in the nucleoid's volume. The compaction is anisotropic, being higher along the long axes of the cell at low crowding levels. At higher crowding levels, the nucleoid becomes spherical, and its compressibility decreases significantly. Furthermore, we find that the compressibility of the nucleoid is not significantly affected by cell growth rates and by prior treatment with rifampicin. The latter results point out that in addition to poly ribosomes, soluble cytoplasmic proteins have a significant contribution in determining the size of the nucleoid. The contribution of poly ribosomes dominates at faster and soluble proteins at slower growth rates.     

Effects of Macromolecular Crowding on Protein Conformational Changes

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.     

Protein self-association induced by macromolecular crowding: a quantitative analysis by magnetic relaxation dispersion

In the presence of high concentrations of inert macromolecules, the self-association of proteins is strongly enhanced through an entropic, excluded-volume effect variously called macromolecular crowding or depletion attraction. Despite the predicted large magnitude of this universal effect and its far-reaching biological implications, few experimental studies of macromolecular crowding have been reported. Here, we introduce a powerful new technique, fast field-cycling magnetic relaxation dispersion, for investigating crowding effects on protein self-association equilibria. By recording the solvent proton spin relaxation rate over a wide range of magnetic field strengths, we determine the populations of coexisting monomers and decamers of bovine pancreatic trypsin inhibitor in the presence of dextran up to a macromolecular volume fraction of 27%. Already at a dextran volume fraction of 14%, we find a 30-fold increase of the decamer population and 510(5)-fold increase of the association constant. The analysis of these results, in terms of a statistical-mechanical model that incorporates polymer flexibility as well as the excluded volume of the protein, shows that the dramatic enhancement of bovine pancreatic trypsin inhibitor self-association can be quantitatively rationalized in terms of hard repulsive interactions.     

Coarse-grained molecular simulation of diffusion and reaction kinetics in a crowded virtual cytoplasm

We present a general-purpose model for biomolecular simulations at the molecular level that incorporates stochasticity, spatial dependence, and volume exclusion, using diffusing and reacting particles with physical dimensions. To validate the model, we first established the formal relationship between the microscopic model parameters (timestep, move length, and reaction probabilities) and the macroscopic coefficients for diffusion and reaction rate. We then compared simulation results with Smoluchowski theory for diffusion-limited irreversible reactions and the best available approximation for diffusion-influenced reversible reactions. To simulate the volumetric effects of a crowded intracellular environment, we created a virtual cytoplasm composed of a heterogeneous population of particles diffusing at rates appropriate to their size. The particle-size distribution was estimated from the relative abundance, mass, and stoichiometries of protein complexes using an experimentally derived proteome catalog from Escherichia coli K12. Simulated diffusion constants exhibited anomalous behavior as a function of time and crowding. Although significant, the volumetric impact of crowding on diffusion cannot fully account for retarded protein mobility in vivo, suggesting that other biophysical factors are at play. The simulated effect of crowding on barnase-barstar dimerization, an experimentally characterized example of a bimolecular association reaction, reveals a biphasic time course, indicating that crowding exerts different effects over different timescales. These observations illustrate that quantitative realism in biosimulation will depend to some extent on mesoscale phenomena that are not currently well understood.     

Molecular crowding affects diffusion and binding of nuclear proteins in heterochromatin and reveals the fractal organization of chromatin

The nucleus of eukaryotes is organized into functional compartments, the two most prominent being heterochromatin and nucleoli. These structures are highly enriched in DNA, proteins or RNA, and thus thought to be crowded. In vitro, molecular crowding induces volume exclusion, hinders diffusion and enhances association, but whether these effects are relevant in vivo remains unclear. Here, we establish that volume exclusion and diffusive hindrance occur in dense nuclear compartments by probing the diffusive behaviour of inert fluorescent tracers in living cells. We also demonstrate that chromatin‐interacting proteins remain transiently trapped in heterochromatin due to crowding induced enhanced affinity. The kinetic signatures of these crowding consequences allow us to derive a fractal model of chromatin organization, which explains why the dynamics of soluble nuclear proteins are affected independently of their size. This model further shows that the fractal architecture differs between heterochromatin and euchromatin, and predicts that chromatin proteins use different target‐search strategies in the two compartments. We propose that fractal crowding is a fundamental principle of nuclear organization, particularly of heterochromatin maintenance.     

Aspects of protein–DNA interactions: a review of quantitative thermodynamic theory for modelling synthetic circuits utilising LacI and CI repressors,IPTG and the reporter gene <Emphasis Type="Italic">lacZ</Emphasis>

Protein–DNA interactions are central to the control of gene expression across all forms of life. The development of approaches to rigorously model such interactions has often been hindered both by a lack of quantitative binding data and by the difficulty in accounting for parameters relevant to the intracellular situation, such as DNA looping and thermodynamic non-ideality. Here, we review these considerations by developing a thermodynamically based mathematical model that attempts to simulate the functioning of an Escherichia coli expression system incorporating two of the best characterised prokaryotic DNA binding proteins, Lac repressor and lambda CI repressor. The key aim was to reproduce experimentally observed reporter gene activities arising from the expression of either wild-type CI repressor or one of three positive-control CI mutants. The model considers the role of several potentially important, but sometimes neglected, biochemical features, including DNA looping, macromolecular crowding and non-specific binding, and allowed us to obtain association constants for the binding of CI and its variants to a specific operator sequence.