Effects of crowding by mono-, di-, and tetrasaccharides on cytochrome c-cytochrome c peroxidase binding: comparing experiment to theory

In cells, protein-protein interactions occur in an environment that is crowded with other molecules, but in vitro studies are almost exclusively performed in dilute solution. To gain information about the effects of crowding on protein complex formation, we used isothermal titration calorimetry to measure the stoichiometry, the free energy change, and the enthalpy change for the binding of yeast iso-1-ferricytochrome c to yeast ferricytochrome c peroxidase in dilute solution and in solutions crowded with the sugars glucose, sucrose, and stachyose. The stoichiometry is 1:1 under all conditions. The sugars stabilize the complex, but by only 0.1-0.5 kcal.mol(-)(1), and the increased stability is not correlated with the change in enthalpy of complex formation. We then compared the measured stability changes to values obtained from several analyses that are currently used to predict crowding-induced changes in biomolecular equilibria. None of the analyses are completely successful by themselves, and the results suggest that a complete analysis must account for both excluded-volume and chemical interactions.     

Simulation and modeling of crowding effects on the thermodynamic and kinetic properties of proteins with atomic details

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.     

Recombinant Escherichia coli GMP reductase: kinetic, catalytic and chemical mechanisms, and thermodynamics of enzyme-ligand binary complex formation

Guanosine monophosphate (GMP) reductase catalyzes the reductive deamination of GMP to inosine monophosphate (IMP). GMP reductase plays an important role in the conversion of nucleoside and nucleotide derivatives of guanine to adenine nucleotides. In addition, as a member of the purine salvage pathway, it also participates in the reutilization of free intracellular bases. Here we present cloning, expression and purification of Escherichia coli guaC-encoded GMP reductase to determine its kinetic mechanism, as well as chemical and thermodynamic features of this reaction. Initial velocity studies and isothermal titration calorimetry demonstrated that GMP reductase follows an ordered bi-bi kinetic mechanism, in which GMP binds first to the enzyme followed by NADPH binding, and NADP(+) dissociates first followed by IMP release. The isothermal titration calorimetry also showed that GMP and IMP binding are thermodynamically favorable processes. The pH-rate profiles showed groups with apparent pK values of 6.6 and 9.6 involved in catalysis, and pK values of 7.1 and 8.6 important to GMP binding, and a pK value of 6.2 important for NADPH binding. Primary deuterium kinetic isotope effects demonstrated that hydride transfer contributes to the rate-limiting step, whereas solvent kinetic isotope effects arise from a single protonic site that plays a modest role in catalysis. Multiple isotope effects suggest that protonation and hydride transfer steps take place in the same transition state, lending support to a concerted mechanism. Pre-steady-state kinetic data suggest that product release does not contribute to the rate-limiting step of the reaction catalyzed by E. coli GMP reductase.     

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.     

Calorimetry of enzyme-catalyzed reactions

This mini-review shows the valuable contributions of Professor Julian Sturtevant to the current applications of calorimetry to the study of enzyme-catalyzed reactions. The more recent applications of calorimetric techniques such as isothermal titration calorimetry and flow calorimetry to the study of enzyme kinetics, as well as the advantages on using calorimetric techniques in the determination of kinetic parameters of enzymes, is also discussed here.     

Effect of molecular crowding on DNA polymerase activity

Live cells contain high concentrations of macromolecules, but almost all experimental biochemical data have been generated from dilute solutions that do not reflect conditions in vivo. To understand biomolecular behavior in vivo, properties studied in vitro are extrapolated to conditions in vivo; however, the molecular conditions within live cells are inherently crowded. The present study investigates the effect of molecular crowding on DNA polymerase activity using polyethylene glycol PEG of various molecular weights as a crowding agent. Polymerase activity assays under various conditions demonstrated that the activities of T7 and Taq DNA polymerases depend on the molecular weight and concentration of the crowding agent. Furthermore, equilibrium and kinetic analyses demonstrated that the binding affinity and catalytic activity of the polymerase increase and decrease, respectively, with increasing PEG concentrations. Based on quantitative parameters of the polymerase reactions, we improved the efficiency of PCR amplification under conditions of molecular crowding. These results suggest that quantitative measurements of biomolecular structure and function are useful for understanding the behavior of biomolecules in vivo and for biotechnology applications in vitro.     

Tactile stimuli perceived by the antennae cause the isolated females to produce gregarious offspring in the desert locust, Schistocerca gregaria

Maternal determination of progeny body size and coloration in the desert locust, Schistocerca gregaria, depends on the crowding conditions experienced during the short sensitive period that occurs two to six days before the deposition of the egg pod. Solitarious (isolated-reared) females produce relatively small eggs that yield solitarious green hatchlings but, females that are exposed to crowded conditions during the sensitive period, produce larger eggs that yield the dark-colored hatchlings characteristic of gregarious forms. The present study aimed to determine the stimuli influencing the maternal determination of progeny characteristics as well as the site at which such stimuli are perceived. By exposing isolated female adults to various combinations of visual, olfactory and tactile stimuli from a crowd of other adults, we found that no crowding effects could be elicited without tactile stimulation. Coating of various body surfaces with nail polish followed by exposure to crowding stimulation suggested that female adults perceive crowding stimuli with their antennae. This finding was supported by another experiment in which the antennae were either removed or covered with wax before the isolated females were exposed to crowded conditions. Neither serotonin nor an antagonist of its receptor affected the density-dependent maternal determination of progeny characteristics when injected into isolated or crowded female adults.     

A natural and readily available crowding agent: NMR studies of proteins in hen egg white

In vitro studies of biological macromolecules are usually performed in dilute, buffered solutions containing one or just a few different biological macromolecules. Under these conditions, the interactions among molecules are diffusion limited. On the contrary, in living systems, macromolecules of a given type are surrounded by many others, at very high total concentrations. In the last few years, there has been an increasing effort to study biological macromolecules directly in natural crowded environments, as in intact bacterial cells or by mimicking natural crowding by adding proteins, polysaccharides, or even synthetic polymers. Here, we propose the use of hen egg white (HEW) as a simple natural medium, with all features of the media of crowded cells, that could be used by any researcher without difficulty and inexpensively. We present a study of the stability and dynamics behavior of model proteins in HEW, chosen as a prototypical, readily accessible natural medium that can mimic cytosol. We show that two typical globular proteins, dissolved in HEW, give NMR spectra very similar to those obtained in dilute buffers, although dynamic parameters are clearly affected by the crowded medium. The thermal stability of one of these proteins, measured in a range comprising both heat and cold denaturation, is also similar to that in buffer. Our data open new possibilities to the study of proteins in natural crowded media.     

Genetic variation of density responses in relation to wing polymorphism in the oriental chinch bug,Cavelerius saccharivorus Okajima (Heteroptera: Lygaeidae)

Responses to nymphal density in the determination of wing form were compared between the offspring from brachypter x brachypter crosses and those from macropter x macropter crosses of the oriental chinch bug, Cavelerius saccharivorus. In the offspring from crosses between macropters, there was a strong tendency for macropters to increase to a rather high level with increasing nymphal density in both sexes. In contrast to this, in the offspring from crosses between brachypters, the appearance rate of macropters attained a maximum value in moderately crowded conditions and conversely decreased to a lower level in more crowded conditions in both sexes. Thus density response patterns concerning the determination of wing form were quite different between the offspring from different crosses in the wing form, indicating that there is a genetic basis underlying wing polymorphism in this species. As for the body size of emerged adults, macropters tended to be larger than brachypters in the same crowded condition. Moreover, the rate of decrease of body size with nymphal density was lower in the offspring from crosses between macropters than in the offspring from crosses between brachypters. This indicated that the former offspring are more tolerant of nymphal crowding than the latter. The difference in such a tolerance against nymphal crowding between the offspring from different crosses was considered to be related to the difference in the appearance of macropters in the crowded conditions between them.     

Calorimetric demonstration of the potential of molecular crowding to emulate the effect of an allosteric activator on pyruvate kinase kinetics

A method based on isothermal calorimetry is described for the direct kinetic assay of pyruvate kinase. In agreement with earlier findings based on the standard coupled assay system for this enzyme in the presence of a fixed ADP concentration, the essentially rectangular hyperbolic dependence of initial velocity upon phosphoenolpyruvate concentration is rendered sigmoidal by the allosteric inhibitor phenylalanine. This effect of phenylalanine can be countered by including a high concentration of a space-filling osmolyte such as proline in the reaction mixtures. This investigation thus affords a dramatic example that illustrates the need to consider potential consequences of thermodynamic nonideality on the kinetics of enzyme reactions in crowded molecular environments such as the cell cytoplasm.     

Structure-function analysis of cytochromes P450 2B

In the last 4 years, breakthroughs were made in the field of P450 2B (CYP2B) structure-function through determination of one ligand-free and two inhibitor-bound X-ray crystal structures of CYP2B4, which revealed many of the structural features required for binding ligands of different size and shape. Large conformational changes of several plastic regions of CYP2B4 can dramatically reshape the active site of the enzyme to fit the size and shape of the bound ligand without perturbing the overall P450 fold. Solution biophysical studies using isothermal titration calorimetry (ITC) have revealed the large difference in the thermodynamic parameters of CYP2B4 in binding inhibitors of different ring chemistry and side chains. Other studies have revealed that the effects of site-specific mutations on steady-state kinetic parameters and mechanism-based inactivation are often substrate dependent. These findings agree with the structural data that the enzymes adopt different conformations to bind various ligands. Thus, the substrate specificity of an individual enzyme is determined not only by active site residues but also non-active site residues that modulate conformational changes that are important for substrate access and rearrangement of the active site to accommodate the bound substrate.     

Glyceraldehyde-3-phosphate dehydrogenase

Conflicting experimental evidence of the pathway of catalysis for the enzyme from rabbit, pig and lobster muscle tissues is reviewed. Transient kinetic studies with the enzyme from rabbit muscle are presented. The results are shown to be consistent with the double-displacement mechanism of catalysis originally proposed by Segal & Boyer (1953). The rate constant for combination of the aldehyde form of the substrate with the NAD+ complex of the enzyme is about 3 X 10(7) M-1 S-1, and for all four subunits of the molecule the rate constant for hydride transfer in the ternary complex formed is greater than 10(3) S-1, consistent with their simultaneous participation in catalysis. Recent steady-state kinetic studies with the rabbit muscle enzyme, in contrast to earlier studies, also provide evidence to support the Segal-Boyer pathway if the kinetic effects of the negative cooperativity of NAD+ binding are taken into account. Experimental data for the binding of NAD+ to the enzyme from muscles and from Bacillus stearothermophilus, and their interpretations, are also briefly reviewed. The information currently available from X-ray crystallography regarding the structures of holoenzyme and apoenzyme from B. stearothermophilus and lobster muscle is outlined.     

Modulation of Calmodulin Plasticity by the Effect of Macromolecular Crowding

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 enhance the population of compact structures, were then confirmed in experimental measurements using fluorescence resonance energy transfer techniques of donor- and acceptor-labeled CaM under normal and crowded conditions. Using protein reconstruction methods, we further explored the folding-energy landscape and examined the structural characteristics of CaM at free-energy basins. 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, 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.     

Protein folding in confined and crowded environments

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 10k_BT 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.     

Structure–function analysis of cytochromes P450 2B

In the last 4 years, breakthroughs were made in the field of P450 2B (CYP2B) structure–function through determination of one ligand-free and two inhibitor-bound X-ray crystal structures of CYP2B4, which revealed many of the structural features required for binding ligands of different size and shape. Large conformational changes of several plastic regions of CYP2B4 can dramatically reshape the active site of the enzyme to fit the size and shape of the bound ligand without perturbing the overall P450 fold. Solution biophysical studies using isothermal titration calorimetry (ITC) have revealed the large difference in the thermodynamic parameters of CYP2B4 in binding inhibitors of different ring chemistry and side chains. Other studies have revealed that the effects of site-specific mutations on steady-state kinetic parameters and mechanism-based inactivation are often substrate dependent. These findings agree with the structural data that the enzymes adopt different conformations to bind various ligands. Thus, the substrate specificity of an individual enzyme is determined not only by active site residues but also non-active site residues that modulate conformational changes that are important for substrate access and rearrangement of the active site to accommodate the bound substrate.     

Three-dimensional stochastic off-lattice model of binding chemistry in crowded environments

Molecular crowding is one of the characteristic features of the intracellular environment, defined by a dense mixture of varying kinds of proteins and other molecules. Interaction with these molecules significantly alters the rates and equilibria of chemical reactions in the crowded environment. Numerous fundamental activities of a living cell are strongly influenced by the crowding effect, such as protein folding, protein assembly and disassembly, enzyme activity, and signal transduction. Quantitatively predicting how crowding will affect any particular process is, however, a very challenging problem because many physical and chemical parameters act synergistically in ways that defy easy analysis. To build a more realistic model for this problem, we extend a prior stochastic off-lattice model from two-dimensional (2D) to three-dimensional (3D) space and examine how the 3D results compare to those found in 2D. We show that both models exhibit qualitatively similar crowding effects and similar parameter dependence, particularly with respect to a set of parameters previously shown to act linearly on total reaction equilibrium. There are quantitative differences between 2D and 3D models, although with a generally gradual nonlinear interpolation as a system is extended from 2D to 3D. However, the additional freedom of movement allowed to particles as thickness of the simulation box increases can produce significant quantitative change as a system moves from 2D to 3D. Simulation results over broader parameter ranges further show that the impact of molecular crowding is highly dependent on the specific reaction system examined.     

Effects of thermodynamic nonideality on the kinetics of ester hydrolysis by alpha-chymotrypsin: a model system with preexistence of the isomerization equilibrium

The effects of a small inert solute, sucrose, on the kinetics of hydrolysis of N-acetyl-tryptophan ethyl ester by bovine alpha-chymotrypsin have been investigated. In studies at pH 7 and 20 degrees C the presence of 0.5 M sucrose in assay mixtures caused no discernible change in kinetic parameters, a result consistent with existence of the enzyme in a single conformational state under those conditions. However, at pH 3.5 and 50 degrees C, conditions under which the enzyme comprises an equilibrium mixture of compact and expanded isomeric states, inclusion of the inert solute led to a considerable decrease in Michaelis constant (0.84 to 0.61 mM) but no significant change in maximal velocity. These results were shown to be amenable to quantitative interpretation in terms of thermodynamic nonideality effects on catalysis by an enzyme undergoing reversible isomerization in the absence of substrate. For that analysis, which required experimental estimates of the equilibrium constant for preexisting isomerization of enzyme and the activity coefficient of substrate, the magnitude of the former (0.3) was obtained by difference spectroscopy: liquid-liquid partition studies with bromobenzene as organic phase were used to determine the effect of sucrose on the activity coefficient of N-acetyltryptophan ethyl ester. Such agreement between experimental kinetic findings and theoretical predictions based on considerations of excluded volume points to the possible use of the space-filling effects of small solutes for delineating the gross extent of conformational changes associated with reversible isomerization of proteins, and hence to the potential of thermodynamic nonideality as a probe for studying protein denaturation mechanisms as well as substrate-mediated changes associated with enzyme reaction mechanisms.     

Macromolecular crowding extends the range of conditions under which DNA polymerase is functional

The nick-translation reaction of E. coli DNA polymerase I (Pol I) was used as a model system to demonstrate the ability of macromolecular crowding to alter the response of an enzyme to a number of basic parameters, such as pH, temperature or inhibitors. In the presence of high concentrations of non-specific polymers, nick translation occurred under a variety of otherwise strongly inhibitory conditions. The conditions tested included a range of pH values or temperatures or inhibitory concentrations of urea, formamide or ethidium bromide. These crowding effects are accentuated at higher ionic strengths, suggesting their origin in increased binding between the polymerase and its DNA template-primer under crowded conditions. Kinetic measurements were consistent with such a mechanism.     

Density-related sterility in Eretmocerus mundus

While studying the reproductive capacity of Eretmocerus mundus Mercet (Hymenoptera: Aphelinidae) we found that varying numbers of females were sterile. Investigations showed that sterility occurred also in field populations, but at a very low rate. Laboratory sterility was significantly correlated with crowding of the parental females during oviposition. Whitefly hosts from which fertile of sterile females emerged did not differ in size, neither did the hind tibiae of fertile females differ in dimensions from those of sterile ones. Behavior of sterile females differed from that of the fertile ones in several parameters. They exhibited less leg drumming, used the ovipositor more frequently and for shorter durations, and changed more readily from probing to host stinging, and from a number of activities to walking. Altogether, their behavior appeared more restless and caused them to contact more hosts than fertile females. The possibility that the sterility is caused by crowding alone, or by the activity of microorganisms acting under crowded conditions, and the merits of the phenomenon for biological pest control are discussed.