Folding of subtilisin BPN': characterization of a folding intermediate

摘要: Subtilisin BPN', an extracellular serine protease from Bacillus amyloliquefaciens, requires a 77 amino acid pro-sequence for correct folding in vivo. We report the observation of metastable intermediate during refolding wild-type and proteolytically inactive mutant subtilisin BPN' that lack pro-sequence. The addition as separate polypeptide chain results to native state. state is stable at different temperatures, pH values, salt concentrations more than week retains its competence folding. possesses compactness between unfolded states. Although it has native-like secondary structure, shows no distinct near-UV CD spectrum strongly reduced dispersion amide methyl regions 1H NMR spectrum. These indicate considerably less tertiary structure possessed by However, conformation structure: high-affinity calcium binding site and, after first noncooperative transition, unfolds with guanidinium chloride cooperative process. support mechanism comprises high energy transition state, which lowered interaction similarity alpha-lytic supports hypothesis common been developed through convergent evolution.