Small effects of amino acid replacements on the reduced and unfolded state of pancreatic trypsin inhibitor.

Abstract

The effects of amino acid replacements on the hydrodynamic volume of reduced and unfolded bovine pancreatic trypsin inhibitor (BPTI) have been examined by gel electrophoresis. The electrophoretic mobilities of the reduced forms of 46 BPTI variants were compared at room temperature in the absence of denaturants. The single substitutions examined include many different types of replacements at sites throughout the polypeptide, and, collectively, alter 22 of the 58 residues of the wild-type protein. The only substitutions found to alter the electrophoretic mobility of the reduced protein by more than approximately 3% are those that change the net charge of the protein. For nine mutants, the rates of disulfide formation in the reduced protein were also examined and found to be very similar to that of the wild-type protein. These results suggest that any structure that may be present in the reduced protein is either relatively insensitive to amino acid replacements or does not greatly influence the averaged properties of the polypeptide chain.