Folding of omega-conotoxins. 2. Influence of precursor sequences and protein disulfide isomerase.
The peptide Ca2+ channel antagonists found in the venoms of Conus snails, omega-conotoxins, are synthesized as precursors that include a leader peptide, presumed to direct the polypeptide to the endoplasmic reticulum, and a propeptide of unknown function. In addition, the precursors are synthesized with a C-terminal Gly residue that is posttranslationally converted to a terminal amide group. In order to determine whether the precursor sequences contain information that helps direct folding 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-terminal Gly residue, and omega-MVIIA-Gly, which differs from the mature form only at the C-terminus. The three forms folded with similar kinetics, 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 brain synaptosomes was approximately 10-fold lower than that of the mature peptide, and the N-terminal propeptide of pro-omega-MVIIA-Gly was found to decrease binding further, by approximately 100-fold. These results suggest that the omega-conotoxins do not rely on the propeptide region of their precursors to facilitate folding. Rather, the mature sequence contains most of the information required to specify the native disulfide pairings and three-dimensional conformation. The C-terminal Gly may enhance the folding efficiency by forming interactions that stabilize the native conformation with respect to other disulfide-bonded forms.
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; D002851:Chromatography, High Pressure Liquid; D007535:Isomerases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D008978:Mollusk Venoms / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D019704:Protein Disulfide-Isomerases; D012908:Snails; D020866:omega-Conotoxin GVIA; D020960:omega-Conotoxins
D002121:Calcium Channel Blockers; D008978:Mollusk Venoms; D010455:Peptides; D020960:omega-Conotoxins; C078452:ziconotide; D020866:omega-Conotoxin GVIA; D007535:Isomerases; D019704:Protein Disulfide-Isomerases