Protein−protein interactions are usually studied in dilute buffered solutions with macromolecule concentrations of <10 g/L. In cells, however, the macromolecule concentration can exceed 300 g/L, resulting in nonspecific interactions between macromolecules. These interactions can be divided into hard-core steric repulsions and "soft" chemical interactions. Here, we test a hypothesis from scaled particle theory; the influence of hard-core repulsions on a protein dimer depends on its shape. We tested the idea using a side-by-side dumbbell-shaped dimer and a domain-swapped ellipsoidal dimer. Both dimers are variants of the B1 domain of protein G and differ by only three residues. The results from the relatively inert synthetic polymer crowding molecules, Ficoll and PEG, support the hypothesis, indicating that the domain-swapped dimer is stabilized by hard-core repulsions while the side-by-side dimer shows little to no stabilization. We also show that protein cosolutes, which interact primarily through nonspecific chemical interactions, have the same small effect on both dimers. Our results suggest that the shape of the protein dimer determines the influence of hard-core repulsions, providing cells with a mechanism for regulating protein−protein interactions. macromolecular crowding | protein−protein interactions | scaled particle theory P rotein−protein interactions are essential for maintaining cellular homeostasis (1). Details of their equilibria under thermodynamically ideal conditions have provided a trove of information. Ideality in this sense refers to dilute solutions, where each monomer contacts only solvent or another monomer, conditions far removed from those in cells where protein− protein interactions evolved. In the cytoplasm, and other cellular compartments and biological fluids, macromolecules can occupy up to 30% of the volume, and their concentrations often exceed 300 g/L (2).Protein molecules take part in more-complex interactions under nonideal conditions. The surrounding macromolecules influence proteins in two ways, neither of which is significant in dilute solution. Hard-core repulsions arise from high volume occupancy, because two molecules cannot occupy the same space at the same time. This volume exclusion favors the most compact state of a protein (3). Chemical interactions comprise transient contacts between protein surfaces arising from the diverse chemical landscapes of proteins (4). When repulsive (i.e., like charges), they favor the state that maximizes the distance between charges, adding to the hard-core repulsions and stabilizing the native state. Attractive chemical interactions (e.g., opposite charges, hydrogen bonds) are destabilizing. We are beginning to understand how hard-core repulsions and chemical interactions affect protein stability (5), but there are few studies about crowding effects on protein−protein interactions (6-9).Given the existential roles of both protein−protein interactions and crowding in biology (10), we are undertaking efforts to determine the effect of cro...
Long accepted as the most important interaction, recent work shows that steric repulsions alone cannot explain the effects of macromolecular cosolutes on the equilibrium thermodynamics of protein stability. Instead, chemical interactions have been shown to modulate, and even dominate, crowding-induced steric repulsions. Here, we use19F NMR to examine the effects of small and large cosolutes on the kinetics of protein folding and unfolding using the metastable 7 kDa N-terminal SH3 domain of the Drosophila signaling protein drk (SH3), which folds by a two-state mechanism. The small cosolutes consist of trimethylamine N-oxide and sucrose, which increase equilibrium protein stability, and urea, which destabilizes proteins. The macromolecules comprise the stabilizing sucrose polymer, Ficoll, and the destabilizing globular protein, lysozyme. We assessed the effects of these cosolutes on the differences in free energy between the folded state and the transition state and between the unfolded ensemble and the transition state. We then examined the temperature dependence to assess changes in activation enthalpy and entropy. The enthalpically mediated effects are more complicated than suggested by equilibrium measurements. We also observed enthalpic effects with the supposedly inert sucrose polymer, Ficoll, that arise from its macromolecular nature. Assessment of activation entropies shows important contributions from solvent and cosolute, in addition to the configurational entropy of the protein that, again, cannot be gleaned from equilibrium data. Comparing the effects of Ficoll to those of the more physiologically relevant cosolute lysozyme reveals that synthetic polymers are not appropriate models for understanding the kinetics of protein folding in cells.
Protein–protein interactions are fundamental to biology yet are rarely studied under physiologically relevant conditions where the concentration of macromolecules can exceed 300 g/L. These high concentrations cause cosolute–complex contacts that are absent in dilute buffer. Understanding such interactions is important because they organize the cellular interior. We used 19F nuclear magnetic resonance, the dimer-forming A34F variant of the model protein GB1, and the cosolutes bovine serum albumin (BSA) and lysozyme to assess the effects of repulsive and attractive charge–charge dimer–cosolute interactions on dimer stability. The interactions were also manipulated via charge-change variants and by changing the pH. Charge–charge repulsions between BSA and GB1 stabilize the dimer, and the effects of lysozyme indicate a role for attractive interactions. The data show that chemical interactions can regulate the strength of protein–protein interactions under physiologically relevant crowded conditions and suggest a mechanism for tuning the equilibrium thermodynamics of protein–protein interactions in cells.
Protein enzymes are the main catalysts in the crowded and complex cellular interior, but their activity is almost always studied in dilute buffered solutions. Studies that attempt to recreate the cellular interior in vitro often utilize synthetic polymers as crowding agents. Here, we report the effects of the synthetic polymer cosolutes Ficoll, dextran, and polyvinylpyrrolidone, and their respective monomers, sucrose, glucose, and 1-ethyl-2-pyrrolidone, on the activity of the 18-kDa monomeric enzyme, Escherichia coli dihydrofolate reductase. At low concentrations, reductase activity increases relative to buffer and monomers, suggesting a macromolecular effect. However, the effect decreases at higher concentrations, approaching, and, in some cases, falling below buffer values. We also assessed activity in terms of volume occupancy, viscosity, and the overlap concentration (where polymers form an interwoven mesh). The trends vary with polymer family, but changes in activity are within threefold of buffer values. We also compiled and analyzed results from previous studies and conclude that alterations of steady-state enzyme kinetics in solutions crowded with synthetic polymers are idiosyncratic with respect to the crowding agent and enzyme.
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