Unfolding and refolding of cardiotoxin III elucidated by reversible conversion of the native and scrambled species

Cardiotoxin analogue III (CTX III) isolated from the venom of the Taiwan Cobra (Naja naja atra) is a small molecular weight, all beta-sheet protein, cross-linked by four disulfide bridges. The unfolding and refolding mechanisms of CTX III have been examined by monitoring the reversible conversion of the native and scrambled species. It is found that, in the presence of a denaturant (urea/guanidinium hydrochloride) and a thiol catalyst, CTX III forms a mixture of scrambled species by shuffling its four native disulfide bonds. Complete unfolding of CTX III can be achieved using either 3.0-4.0 M guanidinium hydrochloride (GdmCl) or 5.0-6.0 M urea. It is observed that GdmCl is thermodynamically more potent but kinetically less efficient than urea in unfolding CTX III. The rate constants of unfolding of CTX III in 8 M urea are significantly greater than that obtained in 5.0 M GdmCl and 8.0 M GdmCl. Interestingly, upon removal of the denaturant, scrambled species of CTX III is found to refold spontaneously through dynamic reshuffling of the non-native disulfides to attain the native disulfide linkages. In addition, CTX III contains highly reactive lysines which are modified by trace amounts of cyanate contaminant which exists invariably even in high-grade urea solutions. The reactive lysines of CTX III are modified by cyanate both in the native and unfolded states of the protein. The modification is nonselective, and the modified product is found to consist of highly heterogeneous species. Surprisingly, these heterogeneous species of modified CTX III are observed to display stability and folding/unfolding properties indistinguishable from those of the native CTX III. The knowledge obtained from the present study, on the conditions to convert the scrambled species, could provide useful clues for a rational design for snake venom cardiotoxins with potential therapeutic applications.