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Macromolecular crowding

2006; Elsevier BV; Volume: 16; Issue: 8 Linguagem: Inglês

10.1016/j.cub.2006.03.047

1879-0445

Allen P. Minton,

Enzyme Structure and Function

What is macromolecular crowding? The term ‘macromolecular crowding’ was coined to connote the influence of mutual volume exclusion upon the energetics and transport properties of macromolecules within a crowded, or highly volume-occupied, medium. Volume exclusion? What's that? Because of steric repulsion, no part of any two macromolecules can be in the same place at the same time. That part of the total volume which cannot be occupied by the center of mass of a particular solute species at a particular instant is called the excluded volume, and the part of total volume that may be occupied is called the available volume (Figure 1). As the fraction of volume occupied by macromolecules of a given size increases, the fraction of volume available to an additional macromolecule of comparable size decreases rapidly, and becomes much less than the fraction of volume available to solvent (water). In freshman chemistry we are taught that the reactivity of a solute is proportional to its concentration, or number of molecules of solute per unit total volume. In fact, this is only strictly true in the highly dilute limit. In a highly volume-occupied solution, the reactivity of a test solute species is determined by the number of molecules of that solute per unit of available volume, which is an effective concentration called the thermodynamic activity. Depending upon the size and shape of the test solute species, and the number density and sizes and shapes of all of the macromolecular solute species in the vicinity of the test species — termed background species — the effective concentration or activity of the test species may exceed its actual concentration by as much as several orders of magnitude! Why is crowding relevant to biology? Biochemical rates and equilibria have traditionally been studied in dilute solution, where the consequences of steric repulsion between solutes are generally negligibly small. In contrast, almost all fluid media in biology contain a high total volume fraction of macromolecules. In special cases, a medium consists primarily of a single species of macromolecule — for example, hemoglobin in hemolysate or albumin in blood serum — but more commonly the medium is highly heterogeneous, as in the case of prokaryotic cytoplasm, containing a mixture of proteins, nucleic acids and polysaccharides in varying proportion. Experiments carried out on solutions containing comparable volume fractions of purified proteins or chemically inert polysaccharides have demonstrated that excluded volume effects in such media can result in the alteration of equilibrium and rate constants by up to several orders of magnitude. How does crowding affect biochemical equilibria? Crowding is a consequence of steric repulsion, a destabilizing interaction that increases the total free energy or work content of the system. Equilibrium theory predicts that if the composition of a system can change to minimize the total free energy of that system, it will do so. Thus crowding is expected to shift equilibria toward a state of the system in which excluded volume is minimized. The extent to which a particular macromolecular species excludes volume to its neighbors generally increases with the ratio of surface to volume of that species. Hence crowding exerts a generalized pressure for the reduction of the surface to volume ratio. This is accomplished in two ways. The first is by favoring compact conformations over extended conformations of flexible macromolecules. The second is by favoring both specific macromolecular associations leading to the formation of well-defined oligomeric species, and nonspecific macromolecular associations leading to the formation of large aggregates of native or nonnative species. How does crowding affect biochemical rates? Crowding can affect reaction rates by two distinct mechanisms. The rate of slow reactions is ordinarily limited by the rate with which reactants pass over a free energy barrier identified as a transition state. In the case of slow reactions, this rate is sufficiently low that the transition state may to a good approximation be treated as if it were in equilibrium with reactant(s) and product(s). Because the attractive interactions that stabilize a complex are ordinarily short-ranged, the transition state of an association reaction tends to resemble the association product more closely than it does the fully separated reactants, and hence exclude a volume to its neighbors similar to that of the fully associated product. For this reason, crowding is expected to increase the association rate constant and have little effect on the dissociation rate constant. In the case of very fast reactions, the rate of an association is limited by the rate with which reactants encounter each other. This rate is dominated by translational diffusion, which decreases monotonically with increased crowding due to the presence of an increasing number of obstacles. Thus crowding is expected to accelerate slow association reactions and decelerate fast association reactions. Are all reactions affected equally by crowding? No. One would not expect a reaction to be affected by crowding if it is not accompanied by a significant change in the volume excluded to background solutes. The binding of a small molecule by a macromolecule would thus be essentially unaffected by crowding unless the binding event was linked to a major change in the conformation or the state of association of the macromolecule. On geometric grounds one would not expect crowding by large macromolecules to greatly affect the behavior of small molecules or significantly smaller macromolecules, which can more easily fit into interstices between large molecules. However, both the dynamic and equilibrium behavior of large macromolecules or macromolecular assemblies would be expected to be greatly affected by the presence of high concentrations of smaller macromolecules. How much of the difference between biochemical reactions in vitro and in vivo can be attributed to crowding? The answer to this question depends upon the particular reaction and the microscopic environment in which the reaction is taking place. Early demonstrations of the large effect of crowding on association equilibria and rates were based upon studies of the behavior of mutant and normal hemoglobins in erythrocytes. Hemolysate is a fairly simple fluid containing primarily hemoglobin, and it can be shown that volume exclusion is a dominating factor in this medium. However, in a more complex heterogeneous environment such as cytoplasm, crowding is probably just one of several nonspecific factors affecting reaction rates and equilibria, such as weak nonspecific associations with background molecules or large structures leading to possible sequestration or adsorption of reactants and/or products. Nevertheless it is essential to keep in mind that, in a crowded biological fluid, the effects of volume exclusion will always be present and play an important role in influencing macromolecular structure and function, independent of and in addition to the influences of other types of interactions. The ubiquity of this phenomenon in biological fluids has been compared to that of gravity.