The effects of single amino acid replacements on the stability of the 14-38 disulfide bond in the native form of bovine pancreatic trypsin inhibitor (BPTI) were measured. A total of 17 mutant proteins, with substitutions at one of 7 residues located 5-15 A from the disulfide in the native wild-type protein, were examined. The replacements were found to decrease the thermodynamic stability of the disulfide, as measured by exchange with thiol-disulfide reagents, by 0.6-5 kcal/mol, corresponding to a range of nearly 100 mV in redox potentials. The effects of the substitutions on disulfide stability were roughly correlated with the changes in side-chain volume, suggesting that optimal packing is a major factor in determining the stability of the disulfide in the wild-type protein. With only one exception, the substitutions also led to increases, as large as 50-fold, in the rates of disulfide reduction by dithiothreitol. The increased rates of reduction suggest that at least a fraction of the mutational destabilization of the disulfide is due to strain in the native protein that is relieved in the transition state for reduction. The stability of the disulfide in a peptide corresponding to the segments that are linked together by the 14-38 disulfide in native BPTI was found to be about 5 kcal/mol less than that of the disulfide in the intact wild-type protein. Together, the results with the mutant proteins and the peptide indicate that the stability of the disulfide in the native protein depends on both the local environment of the disulfide and on the ability of the rest of the protein to favor a conformation that promotes disulfide formation. Study holds ProTherm entries: 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299 Extra Details: additive : EDTA(1 mM), amino acid replacements; disulfide stability; BPTI;,thermodynamic stability
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:14 p.m.
|Number of data points||17|
|Proteins||Pancreatic trypsin inhibitor ; Pancreatic trypsin inhibitor ; Pancreatic trypsin inhibitor|
|Assays/Quantities/Protocols||Experimental Assay: ddG_H2O|
|Libraries||Mutations for sequence RPDFCLEPPYTGPCKARIIRYFYNAKAGLCQTFVYGGCRAKRNNFKSAEDCMRTCGGA|