Protein Fibrillation under Crowded Conditions.

Protein amyloid fibrils have widespread implications for human health. Over the last twenty years, fibrillation has been studied using a variety of crowding agents to mimic the packed interior of cells or to probe the mechanisms and pathways of the process. We tabulate and review these results by considering three classes of crowding agent: synthetic polymers, osmolytes and other small molecules, and globular proteins. While some patterns are observable for certain crowding agents, the results are highly variable and often depend on the specific pairing of crowder and fibrillating protein.

Large cosolutes, small cosolutes, and dihydrofolate reductase activity.

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.

Cosolutes, Crowding, and Protein Folding Kinetics.

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 use 19F 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.

Osmotic Shock Induced Protein Destabilization in Living Cells and Its Reversal by Glycine Betaine.

Many organisms can adapt to changes in the solute content of their surroundings (i.e., the osmolarity). Hyperosmotic shock causes water efflux and a concomitant reduction in cell volume, which is countered by the accumulation of osmolytes. This volume reduction increases the crowded nature of the cytoplasm, which is expected to affect protein stability. In contrast to traditional theory, which predicts that more crowded conditions can only increase protein stability, recent work shows that crowding can destabilize proteins through transient attractive interactions. Here, we quantify protein stability in living Escherichia coli cells before and after hyperosmotic shock in the presence and absence of the osmolyte, glycine betaine. The 7-kDa N-terminal src-homology 3 domain of Drosophila signal transduction protein drk is used as the test protein. We find that hyperosmotic shock decreases SH3 stability in cells, consistent with the idea that transient attractive interactions are important under physiologically relevant crowded conditions. The subsequent uptake of glycine betaine returns SH3 to the stability observed without osmotic shock. These results highlight the effect of transient attractive interactions on protein stability in cells and provide a new explanation for why stressed cells accumulate osmolytes.