title: Effects of amino acid replacements on the reductive unfolding kinetics of pancreatic trypsin inhibitor.
abstract: In order to characterize the major transition states in the disulfide-coupled folding pathway of bovine pancreatic trypsin inhibitor (BPTI), the reductive unfolding kinetics of wild-type BPTI and 18 variants with single amino acid replacements were measured in the presence of varying concentrations of dithiothreitol (DTTSHSH). As observed previously for the wild-type protein, unfolding of the mutant proteins was found to proceed through the formation of a native-like two-disulfide intermediate (NSHSH), followed by either direct reduction of this intermediate or intramolecular rearrangement to generate other two-disulfide species that were then reduced further. From the dependence of the rate of disappearance of NSHSH on the concentration of DTTSHSH, the rate constants for the direct and rearrangement mechanisms were estimated. All of the amino acid replacements examined were found to increase both rate constants, with some mutants unfolding as much as 10,000-fold more rapidly than the wild-type protein. The two rate constants were highly correlated by a linear free energy relationship, suggesting that the transition states for the direct and rearrangement mechanisms are very similar in their response to amino acid replacements. These results are consistent with a model in which the two transition states, which are also the major transition states for disulfide-coupled refolding, are extensively unfolded.
authors.affiliation: Department of Biology, University of Utah, Salt Lake City 84112.
authors.forename: J A
authors.initials: JA
authors.lastname: Mendoza
authors.forename: M B
authors.initials: MB
authors.lastname: Jarstfer
authors.forename: D P
authors.initials: DP
authors.lastname: Goldenberg
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D007611:Aprotinin / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D000596:Amino Acids; D011994:Recombinant Proteins; D007611:Aprotinin