Refolding of bovine pancreatic trypsin inhibitor modified at methionine-52

Abstract

The reduced fragment of pancreatic trypsin inhibitor lacking the six C-terminal residues, which is produced by cyanogen bromide cleavage, formed a seemingly random mixture of disulphide bonds under refolding conditions where normal pancreatic trypsin inhibitor refolds correctly and quantitatively. This illustrates the importance of the C-terminal residues in folding of the normal protein, the uniqueness of the normal folded conformation, and the apparently central role in protein folding of long-range interactions between residues distant in the primary structure. The intact polypeptide chain of reduced pancreatic trypsin inhibitor in which the methionine residue normally at position 52 had been converted to homoserine refolded slightly less readily than the normal reduced compound. This was observed to be due to an altered spectrum of single-disulphide intermediates: the normally predominant intermediate with the 30–51 disulphide bond was less stable by about 0.8 kcal/mol relative to the other normal single-disulphide intermediates. The other steps in refolding appeared to be normal, although the refolded protein was observed to be susceptible to an unexplained reaction with iodoacetate.