Implementation of a k/k(0) method to identify long-range structure in transition states during conformational folding/unfolding of proteins.
Publication Type:Journal Article
Source:Structure, Volume 15, Issue 10, p.1178-89 (2007)
Keywords:Alanine, Animals, Arginine, Cattle, Crystallography, X-Ray, Guanidine, Hydrogen-Ion Concentration, Kinetics, Protein Conformation, Protein Denaturation, Protein Folding, Ribonuclease, Pancreatic, Structure-Activity Relationship, Thermodynamics, Valine
<p>A previously introduced kinetic-rate constant (k/k(0)) method, where k and k(0) are the folding (unfolding) rate constants in the mutant and the wild-type forms, respectively, of a protein, has been applied to obtain qualitative information about structure in the transition state ensemble (TSE) of bovine pancreatic ribonuclease A (RNase A), which contains four native disulfide bonds. The method compares the folding (unfolding) kinetics of RNase A, with and without a covalent crosslink and tests whether the crosslinked residues are associated in the folding (unfolding) transition state (TS) of the noncrosslinked version. To confirm that the fifth disulfide bond has not introduced a significant structural perturbation, we solved the crystal structure of the V43C-R85C mutant to 1.6 A resolution. Our findings suggest that residues Val43 and Arg85 are not associated, and that residues Ala4 and Val118 may form nonnative contacts, in the folding (unfolding) TSE of RNase A.</p>