Macromolecular Crowding Effects on Globular Protein Stability.

摘要

Macromolecular crowding inside cells is proposed to change many aspects of proteins compared to dilute solution. As such, it is an increasingly studied topic, both theoretically and experimentally. However, the difficulty of both theoretically modeling the intracellular milieu and manipulating its contents experimentally present roadblocks to a full picture of crowding inside cells. In vitro studies of macromolecular crowding allow us to study the effects of crowding agent identity, size, and concentration on globular protein stability in a highly controllable fashion. I used NMR-detected amide proton exchange to study the effects of poly(vinylpyrrolidone) at varying molecular weights and concentrations on the stability of chymotrypsin inhibitor 2. This residue-level study is the first to reveal both volume exclusion and weak interaction effects as contributors to protein stability in crowded conditions. I also studied the effects of a microgel crowder on the stability and dynamics of chymotrypsin inhibitor 2, displaying an upper limit to the size effect of crowding agents. This study also revealed no link between protein stability and ps-ns timescale backbone dynamics. Amide proton exchange was also used to study the effects of bovine serum albumin and lysozyme as crowding agents on chymotrypsin inhibitor 2. This is the first reported study of protein stability when subjected to crowding by another protein, and provides some important implications for the stability of proteins inside cells.