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Fully oxidized scrambled isomers are essential and predominant folding intermediates of cardiotoxin‐III
Author(s) -
Chang Jui-Yoa,
Lu Bao-Yuan,
Lin Curtis C.-J.,
Yu Chin
Publication year - 2006
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2005.12.064
Subject(s) - oxidative folding , cardiotoxin , chemistry , folding (dsp implementation) , protein disulfide isomerase , thiol , protein folding , glutathione , disulfide bond , stereochemistry , combinatorial chemistry , biochemistry , venom , electrical engineering , enzyme , engineering
Scrambled isomers (X‐isomers) are fully oxidized, non‐native isomers of disulfide proteins. They have been shown to represent important intermediates along the pathway of oxidative folding of numerous disulfide proteins. A simple method to assess whether X‐isomers present as folding intermediate is to conduct oxidative folding of fully reduced protein in the alkaline buffer alone without any supplementing thiol catalyst or redox agent. Cardiotoxin‐III (CTX‐III) contains 60 amino acids and four disulfide bonds. The mechanism of oxidative folding of CTX‐III has been systematically characterized here by analysis of the acid trapped folding intermediates. Folding of CTX‐III was shown to proceed sequentially through 1‐disulfide, 2‐disulfide, 3‐disulfide and 4‐disulfide (scrambled) isomers as folding intermediates to reach the native structure. When folding of CTX‐III was performed in the buffer alone, more than 97% of the protein was trapped as 4‐disulfide X‐isomers, unable to convert to the native structure due to the absence of thiol catalyst. In the presence of thiol catalyst (GSH) or redox agents (GSH/GSSG), the recovery of native CTX‐III was 80–85%. These results demonstrate that X‐isomers play an essential and predominant role in the oxidative folding of CTX‐III.