Volume exclusion and soft interaction effects on protein stability under crowded conditions.

TLDR: The first systematic, quantitative, residue-level study of crowding effects on the equilibrium stability of a globular protein is reported, concluding that the role of native-state binding and other soft interactions needs to be seriously considered when applying both theory and experiment to studies of macromolecular crowding.

Abstract: Most proteins function in nature under crowded conditions, and crowding can change protein properties. Quantification of crowding effects, however, is difficult because solutions containing hundreds of grams of macromolecules per liter often interfere with the observation of the protein being studied. Models for macromolecular crowding tend to focus on the steric effects of crowders, neglecting potential chemical interactions between the crowder and the test protein. Here, we report the first systematic, quantitative, residue-level study of crowding effects on the equilibrium stability of a globular protein. We used a system comprising poly(vinylpyrrolidone)s (PVPs) of varying molecular weights as crowding agents and chymotrypsin inhibitor 2 (CI2) as a small globular test protein. Stability was quantified with NMR-detected amide 1H exchange. We analyzed the data in terms of hard particle exclusion, confinement, and soft interactions. For all crowded conditions, nearly every observed residue experiences a ...