Fully oxidized scrambled isomers are essential and predominant folding intermediates of cardiotoxin-III.
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.
Chang
Jui-Yoa
JY
Research Center for Protein Chemistry, Institute of Molecular Medicine, The University of Texas, Houston, TX 77030, USA. rowen.chang@uth.tmc.edu
Lu
Bao-Yuan
BY
Lin
Curtis C-J
CC
Yu
Chin
C
D000818:Animals; D002021:Buffers; D004179:Cobra Cardiotoxin Proteins / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D004546:Elapid Venoms / Q000737:chemistry; D007536:Isomerism; D008239:Lysine / Q000737:chemistry; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017510:Protein Folding
D002021:Buffers; D004179:Cobra Cardiotoxin Proteins; D004220:Disulfides; D004546:Elapid Venoms; C082202:cardiotoxin III, Naja naja atra; D008239:Lysine