phi-Values for BPTI folding intermediates and implications for transition state analysis

Amino acid replacements were used to probe the roles of 14 sites in two wel l-characterized intermediates in the folding pathway of bovine pancreatic t rypsin inhibitor (BPTI), One of these intermediates contains one of the thr ee disulfides found in the native protein (30-51). NMR studies have shown t hat approximately two-thirds of this polypeptide has a native-like conforma tion. The other intermediate contains two native disulfides (30-51 and 5-55 ) and has a fully folded conformation. The phi -values for a majority of re sidues were <1, indicating that the native protein was significantly more d estabilized than either intermediate even when the altered residue was loca ted in a well-ordered region of the intermediate. These observations sugges t that folding intermediates and transition states may generally be more st ructured than indicated by <phi>-values alone.