FOLDING OF OMEGA-CONOTOXINS .2. INFLUENCE OF PRECURSOR SEQUENCES AND PROTEIN DISULFIDE-ISOMERASE

The peptide Ca2+ channel antagonists found in the venoms of Conus snai ls, omega-conotoxins, are synthesized as precursors that include a lea der peptide, presumed to direct the polypeptide to the endoplasmic ret iculum, and a propeptide of unknown function. In addition, the precurs ors are synthesized with a C-terminal Gly residue that is posttranslat ionally converted to a terminal amide group. In order to determine whe ther the precursor sequences contain information that helps direct fol ding of the mature sequences, the disulfide-coupled folding of mature omega-conotoxin MVIIA was compared with that of two putative precursor forms: pro-omega-MVIIA-Gly, which contains the propeptide and the C-t erminal Gly residue, and omega-MVIIA-Gly, which differs from the matur e form only at the C-terminus. The three forms folded with similar kin etics, but the folding efficiency of omega-MVIIA-Gly was greater than 80%, versus approximately 50% for both mature omega-MVIIA and the form containing the propeptide. The enzyme protein disulfide isomerase was found to catalyze disulfide formation and folding of all three forms similarly. The affinity of omega-MVIIA-Gly for receptors in chick brai n synaptosomes was approximately 10-fold lower than that of the mature peptide, and the N-terminal propeptide of pro-omega-MVIIA-Gly was fou nd to decrease binding further, by approximately 100-fold. These resul ts suggest that the omega-conotoxins do not rely on the propeptide reg ion of their precursors to facilitate folding. Rather, the mature sequ ence contains most of the information required to specify the native d isulfide pairings and three-dimensional conformation. The C-terminal G ly may enhance the folding efficiency by forming interactions that sta bilize the native conformation with respect to other disulfide-bonded forms.