Partially folded conformation of the (30-51) intermediate in the disulphide folding pathway of bovine pancreatic trypsin inhibitor: ¹H and ¹⁵N resonance assignments and determination of backbone dynamics from ¹⁵N relaxation measurements

An analogue of the important folding intermediate of BPTI with only the disulphide bond between Cys30 and Cys51 has been characterized by ¹H and ¹⁵N NMR techniques. In particular, the dynamics of the polypeptide backbone were characterized using (¹H)-¹⁵N NOE and ¹⁵N T₁ and T₂ relaxation data. The intermediate is partially folded, with part of the polypeptide chain stably folded and the remainder flexible or unfolded. The folded portion consists of the major elements of native-like secondary structure interacting through the hydrophobic core of the molecule. The ¹⁵N relaxation data show that the N-terminal 15 residues are very flexible, and the (¹H,¹H) NOESY data show that these residues have no NOE interactions with the remainder of the molecule. The segment of residues 37 to 41 is also flexible. These observations explain why during folding this intermediate most readily forms any of the possible disulphide bonds between Cys5, Cys14 and Cys38, including the non-native 5-14 and 5-38 bonds. The native-like folded portion of the molecule limits the possible disulphide bonds that can be formed to those in the remainder of the polypeptide chain. Also, forming the non-native disulphide bonds need not involve any disruption of that folded structure, as the Cys residues involved are in flexible regions of the molecule.