Impact of reconstituted cytosol on protein stability.
Protein stability is usually studied in simple buffered solutions, but most proteins function inside cells, where the heterogeneous and crowded environment presents a complex, nonideal system. Proteins are expected to behave differently under cellular crowding owing to two types of contacts: hard-core repulsions and weak, chemical interactions. The effect of hard-core repulsions is purely entropic, resulting in volume exclusion owing to the mere presence of the crowders. The weak interactions can be repulsive or attractive, thus enhancing or diminishing the excluded volume, respectively. We used a reductionist approach to assess the effects of intracellular crowding. Escherichia coli cytoplasm was dialyzed, lyophilized, and resuspended at two concentrations. NMR-detected amide proton exchange was then used to quantify the stability of the globular protein chymotrypsin inhibitor 2 (CI2) in these crowded solutions. The cytosol destabilizes CI2, and the destabilization increases with increasing cytosol concentration. This observation shows that the cytoplasm interacts favorably, but nonspecifically, with CI2, and these interactions overcome the stabilizing hard-core repulsions. The effects of the cytosol are even stronger than those of homogeneous protein crowders, reinforcing the biological significance of weak, nonspecific interactions.
Departments of Chemistry and Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599.
Mohona
M
Sarkar
Austin E
AE
Smith
Gary J
GJ
Pielak
D002851:Chromatography, High Pressure Liquid; D003593:Cytoplasm / Q000737:chemistry*; D004926:Escherichia coli; D046911:Macromolecular Substances / Q000032:analysis*; D013058:Mass Spectrometry; D008954:Models, Biological; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry; D060066:Protein Interaction Maps; D055550:Protein Stability / Q000187:drug effects*
D046911:Macromolecular Substances; D010455:Peptides; D010940:Plant Proteins; C093336:chymotrypsin inhibitor 2