Roles of individual disulfide bonds in the stability and folding of an omega-conotoxin.
Although it contains only 25 amino acid residues, omega-conotoxin MVIIA folds into a well-defined three-dimensional structure that is stabilized by 3 disulfide bonds. To assess the contributions of the disulfides to folding and stability, three analogues, each with one pair of disulfide-bonded Cys residues replaced with Ala, were prepared and characterized. The analogues also contained a C-terminal Gly residue that is believed to be present when the peptide folds in vivo and has been shown previously to stabilize the native structure. Circular dichroism spectra and biological assays of the analogues indicated that removing any one of the disulfides greatly destabilized the native conformation. The two disulfides in each analogue were also reduced much more rapidly than in the native form with three disulfides. When the analogues were fully reduced and allowed to form disulfides in the presence of oxidized and reduced glutathione, the native disulfides were not formed in preference to non-native disulfides, further indicating that the forms with two-native disulfides are not significantly stabilized by noncovalent interactions. However, the measured equilibrium constants for disulfide formation indicate that forming any two of the three native disulfides leads to an effective concentration of approximately 25-50 M for the two remaining thiols. The two-disulfide analogues thus appear to represent a stage of folding in which the polypeptide is constrained to a distribution of relatively compact conformations that greatly favor formation of the third disulfide and the final folded structure.
Price-Carter
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Department of Biology, University of Utah, Salt Lake City 84112-0840, USA.
Hull
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Goldenberg
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D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D008978:Mollusk Venoms / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D012908:Snails; D020866:omega-Conotoxin GVIA; D020960:omega-Conotoxins
D004220:Disulfides; D008978:Mollusk Venoms; D010455:Peptides; D020960:omega-Conotoxins; C078452:ziconotide; D020866:omega-Conotoxin GVIA