Folding of omega-conotoxins. 1. Efficient disulfide-coupled folding of mature sequences in vitro.
Disulfide-coupled refolding reactions of five omega-conotoxins, Ca2+ channel antagonists derived from marine snails of the genus Conus, were examined. These peptides are 23-26 amino acid residues long, and the native conformation of each is stabilized by three disulfide bonds. Although the primary structures of the peptides show only limited sequence similarity, the patterns of disulfides and three-dimensional conformations are very similar. Refolding of the reduced proteins was promoted by the disulfide form of glutathione (GSSG) in the presence of reduced glutathione (GSH). All five of the peptides examined were able to refold to the native conformation, as judged by reversed-phase HPLC behavior, with efficiencies of 16% for omega-MVIIC, 28% for omega-MVIID, and 50% for omega-MVIIA, omega-GVIA, and omega-SVIA. The refolded form of omega-MVIIA was further shown to have biological activity indistinguishable from that of the native form, as well as the same rate of reductive unfolding in the presence of dithiothreitol. The overall folding rate and efficiency of omega-MVIIA was found to be quite sensitive to the thiol-disulfide redox potential, with optimum rates and yields obtained in the presence of GSSG and GSH at concentrations similar to those believed to be present in the endoplasmic reticulum. The folding efficiency of this peptide was greatly reduced by the addition of 8 M urea, indicating that formation of the correct disulfides is determined largely by noncovalent interactions, as opposed to steric constraints arising from the spacing between Cys residues. These results demonstrate that the mature forms of at least some omega-conotoxins contain sufficient information to direct correct folding and disulfide formation, in spite of their small size and limited sequence conservation.
Price-Carter
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Department of Biology, University of Utah, Salt Lake City 84112, USA.
Gray
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Goldenberg
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D000595:Amino Acid Sequence; D000818:Animals; D002121:Calcium Channel Blockers / Q000737:chemistry* / Q000378:metabolism; D020916:Conotoxins; D004220:Disulfides / Q000378:metabolism*; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism; D019803:Glutathione Disulfide; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008978:Mollusk Venoms / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012908:Snails; D020866:omega-Conotoxin GVIA; D020960:omega-Conotoxins
D002121:Calcium Channel Blockers; D020916:Conotoxins; D004220:Disulfides; D008978:Mollusk Venoms; D010455:Peptides; D020960:omega-Conotoxins; C077381:omega-conotoxin SVIA; C075851:omega-conotoxin-MVIIC; C085882:omega-conotoxin MVIID; C078452:ziconotide; D020866:omega-Conotoxin GVIA; D005978:Glutathione; D019803:Glutathione Disulfide