Protein behavior in crowded environments (2008)

Undergraduates: Christopher Barnes, Christopher O. Barnes Lisa M. Charlton

Faculty Advisor: Gary Pielak
Department: Chemistry

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Macromolecular crowding has significant thermodynamic and kinetic consequences for biological macromolecules. Experimental evidence has shown that crowding enhances protein association, increases the rate of folding and refolding, and affects diffusion in the cytoplasm. Statistical thermodynamic models predict that macromolecular crowding increases protein stability, yet quantitative, residue-level experimental evidence supporting these predictions is largely absent. Here we report the first residue-specific information about the effects of macromolecular crowding on protein stability. We used NMR-detected amide proton exchange to quantify the effects of 300 g/L polyvinyl pyrrolidone (PVP, 40 kDa) on the stability of the globular protein chymotrypsin inhibitor 2. Crowding increases the equilibrium constant for folding by up to 100-fold compared to dilute solution and the model of the PVP monomer. We also show that, consistent with the local unfolding model, the magnitude of the increase depends on the location of the residue in the structure. Our results demonstrate that the increase in stability upon addition of PVP is the result of macromolecular crowding.