Autotomic behavior of the propeptide in propeptide-mediated folding of prosubtilisin E

The 77 residue propeptide at the N-terminal end of subtilisin E plays an essential role in subtilisin folding as a tailor-made intramolecular chaperone. Upon completion of folding, the propeptide is autoprocessed and removed by subtilisin digestion. This propeptide-mediated protein folding has been used as a paradigm for the study of protein folding. Here, we show by three independent methods, that the propeptide domain and the subtilisin domain show distinctive intrinsic stability that is obligatory for efficient autoprocessing of the propeptide domain. Two tryptophan residues, Trp106 and Trp113, on the surface of subtilisin located on one of the two helices that form the interface between the propeptide and the subtilisin domains play a key role in maintaining the distinctive instability of the propeptide domain, after completion of folding. When either of the Trp residues was substituted with Tyr, the characteristic biphasic heat denaturation profile of two domains unfolding was not observed, resulting in a single transition of denaturation. The results provide evidence that the propeptide not only plays an essential role in subtilisin folding, but upon completion of folding it behaves as an independent domain. Once the propeptide-mediated folding is completed, the propeptide domain is readily eliminated without interference from the subtilisin domain. This "autotomic" behavior of the propeptide may be a prevailing principle in propeptide-mediated protein folding.