COMPARISON OF THE (30-51, 14-38) 2-DISULFIDE FOLDING INTERMEDIATES OFTHE HOMOLOGOUS PROTEINS DENDROTOXIN-K AND BOVINE PANCREATIC TRYPSIN-INHIBITOR BY 2-DIMENSIONAL H-1 NUCLEAR-MAGNETIC-RESONANCE

The disulphide folding pathway of bovine pancreatic trypsin inhibitor (BPTI) revealed that the native conformation is still stable in each i ntermediate state with two native disulphide linkages, in the absence of each of the corresponding third disulphide bends. This is thought t o be a consequence of the extreme stability of the native BPTI conform ation. The current study addresses the question of whether the native- like conformation would be populated significantly at the two-disulphi de stage in disulphide refolding if the final native structure is less stable than in the case of BPTI. Dendrotoxin K from black mamba venom provides a good model to test this, since it contains the BPTI fold a nd was shown to fold predominantly via the same pathway, hut its nativ e conformation is less stable than that of BPTI. The conformation of a chemically trapped two-disulphide intermediate in the disulphide refo lding of dendrotoxin K, with blocking groups on Cys5 and Cys55 and dis ulphide bonds between Cys30 and Cys51, and Cys14 and Cys38, respective ly, has been determined by H-1 NMR spectroscopy and compared to those of the native protein and of the corresponding intermediate in BPTI. T he analysis reveals that the dendrotoxin K intermediate adopts a partl y-folded conformation, in contrast to the quasi-native conformation of the corresponding BPTI intermediate. It is similar to the partly-fold ed conformation of the BPTI intermediate with just the Cys30-Cys51 dis ulphide bond, but with a more fixed conformation in the region of the Cys14-Cys38 disulphide bond. The destabilisation of the fully native c onformation of the dendrotoxin K intermediate, relative to BPTI, appea rs to reduce the cooperativity of the folding process. (C) 1996 Academ ic Press Limited