Mutants of bovine pancreatic trypsin inhibitor lacking cysteines 14 and 38 can fold properly.

It is a generally accepted principle of biology that a protein's primary sequence is the main determinant of its tertiary structure. However, the mechanism by which a protein proceeds from an unfolded, disordered state to a folded, relatively well-ordered, native conformation is obscure. Studies have been initiated to examine the "genetics" of protein folding, with mutants of bovine pancreatic trypsin inhibitor (BPTI) being used to explore the nature of the specific intramolecular interactions that direct this process. Previous work with BPTI chemically modified at cysteines 14 and 38 indicated that transient disulfide bond formation by these residues contributed to efficient folding at 25 degrees C. In the present work, mutants of BPTI in which these cysteines were replaced by alanines or threonines were made and the mutant proteins were produced by a heterologous Escherichia coli expression system. At 25 degrees C in vitro, the refolding behavior of these mutants was characterized by a pronounced lag. However, when expressed at 37 degrees C in E. coli, or when refolded at 37 degrees or 52 degrees C in vitro, the mutant proteins folded readily into the native conformation, albeit at a rate somewhat slower than that exhibited by wild-type BPTI. These results indicate that, at physiological temperatures, BPTI lacking cysteines 14 and 38 can refold quantitatively.