Comparison of the (30-51, 14-38) two-disulphide folding intermediates of the homologous proteins dendrotoxin K and bovine pancreatic trypsin inhibitor by two-dimensional 1H nuclear magnetic resonance.
The disulphide folding pathway of bovine pancreatic trypsin inhibitor (BPTI) revealed that the native conformation is still stable in each intermediate state with two native disulphide linkages, in the absence of each of the corresponding third disulphide bonds. This is thought to be a consequence of the extreme stability of the native BPTI conformation. The current study addresses the question of whether the native-like conformation would be populated significantly at the two-disulphide stage in disulphide refolding if the final 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 and was shown to fold predominantly via the same pathway, but its native conformation is stable than that of BPTI. The conformation of a chemically trapped two-disulphide intermediate in the disulphide refolding of dendrotoxin K, with blocking groups on Cys5 and Cys55 and disulphide bonds between Cys30 and Cys51, and Cys14 and Cys38, respectively, has been determined by 1H NMR spectroscopy and compared to those of the native protein and of the corresponding intermediate in BPTI. The analysis reveals that the dendrotoxin K intermediate adopts a partly-folded conformation, in contrast to the quasi-native conformation of the corresponding BPTI intermediate. It is similar to the partly-folded conformation of the BPTI intermediate with just the Cys30-Cys-51 disulphide bond, but with a more fixed conformation in the region of the Cys14-Cys38 disulphide bond. The destabilisation of the fully native conformation of the dendrotoxin K intermediate, relative to BPTI, appears to reduce the cooperativeity of the folding process.
Kortemme
T
T
European Molecular Biology Laboratory, Heidelberg, Germany.
Hollecker
M
M
Kemmink
J
J
Creighton
T E
TE
D000818:Animals; D002417:Cattle; D009682:Magnetic Resonance Spectroscopy; D010455:Peptides / Q000737:chemistry*; D017510:Protein Folding; D014361:Trypsin Inhibitors / Q000737:chemistry*
D010455:Peptides; D014361:Trypsin Inhibitors; C081506:dendrotoxin K