FOLDING OF OMEGA-CONOTOXINS - EFFICIENT DISULFIDE-COUPLED FOLDING OF MATURE SEQUENCES IN-VITRO

Disulfide-coupled refolding reactions of five omega-conotoxins, Ca2+ c hannel 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. Al though the primary structures of the peptides show only limited sequen ce similarity, the patterns of disulfides and three-dimensional confor mations are very similar. Refolding of the reduced proteins was promot ed by the disulfide form of glutathione (GSSG) in the presence of redu ced glutathione (GSH). All five of the peptides examined were able to refold to the native conformation, as judged by reversed-phase HPLC be havior, with efficiencies of 16% for omega-MVIIC, 28% for omega-MVIID, and 50% for omega-MVIIA, omega-GVIA, and omega-SVIA. The refolded for m of omega-MVIIA was further shown to have biological activity indisti nguishable from that of the native form, as well as the same rate of r eductive unfolding in the presence of dithiothreitol. The overall fold ing 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 foldin g efficiency of this peptide was greatly reduced by the addition of 8 M urea, indicating that formation of the correct disulfides is determi ned largely by noncovalent interactions, as opposed to steric constrai nts arising from the spacing between Cys residues. These results demon strate that the mature forms of at least some omega-conotoxins contain sufficient information to direct correct folding and disulfide format ion, in spite of their small size and limited sequence conservation.