Yaqiang Wang

Macromolecular Crowding and Protein Stability

An understanding of cellular chemistry requires knowledge of how crowded environments affect proteins. The influence of crowding on protein stability arises from two phenomena, hard-core repulsions and soft (i.e., chemical) interactions. Most efforts to understand crowding effects on protein stability, however, focus on hard-core repulsions, which are inherently entropic and stabilizing. We assessed these phenomena by measuring the temperature dependence of NMR-detected amide proton exchange and used these data to extract the entropic and enthalpic contributions of crowding to the stability of ubiquitin. Contrary to expectations, the contribution of chemical interactions is large and in many cases dominates the contribution from hardcore repulsions. Our results show that both chemical interactions and hard-core repulsions must be considered when assessing the effects of crowding and help explain previous observations about protein stability and dynamics in cells.

Protein Crowding Tunes Protein Stability

Thirty percent of a cells volume is filled with macromolecules, and protein chemistry in a crowded environment is predicted to differ from that in dilute solution. We quantified the effect of crowding by globular proteins on the equilibrium thermodynamic stability of a small globular protein. Theory has long predicted that crowding should stabilize proteins, and experiments using synthetic polymers as crowders show such stabilizing effects. We find that protein crowders can be mildly destabilizing. The destabilization arises from a competition between stabilizing excluded-volume effects and destabilizing nonspecific interactions, including electrostatic interactions. This competition results in tunable stability, which could impact our understanding of the spatial and temporal roles of proteins in living systems.

Effects of Proteins on Protein Diffusion

Despite increased attention, little is known about how the crowded intracellular environment affects basic phenomena like protein diffusion. Here, we use NMR to quantify the rotational and translational diffusion of a 7.4-kDa test protein, chymotrypsin inhibitor 2 (CI2), in solutions of glycerol, synthetic polymers, proteins, and cell lysates. As expected, translational diffusion and rotational diffusion decrease with increasing viscosity. In glycerol, for example, the decrease follows the Stokes-Einstein and Stokes-Einstein-Debye laws. Synthetic polymers cause negative deviation from the Stokes laws and affect translation more than rotation. Surprisingly, however, protein crowders have the opposite effect, causing positive deviation and reducing rotational diffusion more than translational diffusion. Indeed, bulk proteins severely attenuate the rotational diffusion of CI2 in crowded protein solutions. Similarly, CI2 diffusion in cell lysates is comparable to its diffusion in crowded protein solutions, supporting the biological relevance of the results. The rotational attenuation is independent of the size and total charge of the crowding protein, suggesting that the effect is general. The difference between the behavior of synthetic polymers and protein crowders suggests that synthetic polymers may not be suitable mimics of the intracellular environment. NMR relaxation data reveal that the source of the difference between synthetic polymers and proteins is the presence of weak interactions between the proteins and CI2. In summary, weak but nonspecific, noncovalent chemical interactions between proteins appear to fundamentally impact protein diffusion in cells.

Protein 19F NMR in Escherichia coli

Although overexpression and (15)N enrichment facilitate the observation of resonances from disordered proteins in Escherichia coli, (15)N enrichment alone is insufficient for detecting most globular proteins. Here, we explain this dichotomy and overcome the problem while extending the capability of in-cell NMR by using (19)F-labeled proteins. Resonances from small (approximately 10 kDa) globular proteins containing the amino acid analogue 3-fluoro-tyrosine can be observed in cells, but for larger proteins the (19)F resonances are broadened beyond detection. Incorporating the amino acid analogue trifluoromethyl-L-phenylalanine allows larger proteins (up to 100 kDa) to be observed in cells. We also show that site-specific structural and dynamic information about both globular and disordered proteins can be obtained inside cells by using (19)F NMR.

Macromolecular Crowding Fails To Fold a Globular Protein in Cells

Proteins perform their functions in cells where macromolecular solutes reach concentrations of >300 g/L and occupy >30% of the volume. The volume excluded by these macromolecules stabilizes globular proteins because the native state occupies less space than the denatured state. Theory predicts that crowding can increase the ratio of folded to unfolded protein by a factor of 100, amounting to 3 kcal/mol of stabilization at room temperature. We tested the idea that volume exclusion dominates the crowding effect in cells using a variant of protein L, a 7 kDa globular protein with seven lysine residues replaced by glutamic acids; 84% of the variant molecules populate the denatured state in dilute buffer at room temperature, compared with 0.1% for the wild-type protein. We then used in-cell NMR spectroscopy to show that the cytoplasm of Escherichia coli does not overcome even this modest (∼1 kcal/mol) free-energy deficit. The data are consistent with the idea that nonspecific interactions between cytoplasmic components can overcome the excluded-volume effect. Evidence for these interactions is provided by the observations that adding simple salts folds the variant in dilute solution but increasing the salt concentration inside E. coli does not fold the protein. Our data are consistent with the results of other studies of protein stability in cells and suggest that stabilizing excluded-volume effects, which must be present under crowded conditions, can be ameliorated by nonspecific interactions between cytoplasmic components.

Translational and rotational diffusion of a small globular protein under crowded conditions.

Protein-protein interaction is the fundamental step of biological signal transduction. Interacting proteins find each other by diffusion. To gain insight into diffusion under the crowded conditions found in cells, we used nuclear magnetic resonance spectroscopy (NMR) to measure the effects of solvent additives on the translational and rotational diffusion of the 7.4 kDa globular protein, chymotrypsin inhibitor 2. The additives were glycerol and the macromolecular crowding agent, polyvinyl pyrrolidone (PVP). Both translational diffusion and rotational diffusion decrease with increasing solution viscosity. For glycerol, the decrease obeys the Stokes-Einstein and Stokes-Einstein Debye laws. Three types of deviation are observed for PVP: the decrease in diffusion with increased viscosity is less than predicted, this negative deviation is greater for rotational diffusion, and the negative deviation increases with increasing PVP molecular weight. We discuss our results in terms of other studies on the effects of macromolecules on globular protein diffusion.