Macromolecular crowding fails to fold a globular protein in cells. J Am Chem Soc 2011 Jun 01;133(21):8082-5
Date
05/04/2011Pubmed ID
21534566Pubmed Central ID
PMC3102155DOI
10.1021/ja201206tScopus ID
2-s2.0-79957787217 (requires institutional sign-in at Scopus site) 138 CitationsAbstract
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.
Author List
Schlesinger AP, Wang Y, Tadeo X, Millet O, Pielak GJAuthor
Yaqiang Wang PhD, MS Assistant Professor in the Biophysics department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Bacterial ProteinsCytoplasm
Macromolecular Substances
Nuclear Magnetic Resonance, Biomolecular
Protein Folding
Protein Stability
