Factors governing selective formation of specific disulfides in synthetic variants of alpha-conotoxin.
alpha-Conotoxin GI is a snail toxin protein consisting of 13 amino acids cross-linked by 2 intramolecular disulfide bridges. This toxin is an antagonist of acetylcholine receptors. The native sequence has been synthesized, along with nine additional variants in which non-cysteine residues are replaced by alanine or the cysteine positions are altered. Each reduced peptide has been oxidized by reaction with oxygen or glutathione both in a folding buffer and in 6 M guanidine hydrochloride. Purified products of oxidation have been characterized with respect to molecular weights and the positions of disulfides. The four cysteines in conotoxin can form two intramolecular disulfides in three different combinations. Relative yields of each of the three isomers have been determined, thereby permitting evaluation of the roles of non-cysteine residues and cysteine placements in the folding of conotoxin. Cysteine positions dominate factors directing formation of the nativelike isomer in a manner that may be predicted from equilibrium constants for loop formation in model peptides containing two cysteines. Alanine substitutions at several positions which are conserved in naturally occurring conotoxins affect the discrimination between the two most favored disulfide arrangements. Substitutions at three nonconserved positions have no structural effect on isomer yields. It therefore is possible to vary these latter three positions in a manner which might help to generate a functional binding surface which is complementary to receptors in the specific prey of a particular species of snail, without affecting the toxin's folding.
Zhang
R M
RM
Department of Biological Sciences, State University of New York, Buffalo 14260.
Snyder
G H
GH
D000595:Amino Acid Sequence; D020916:Conotoxins; D004220:Disulfides / Q000032:analysis; D005978:Glutathione; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D008978:Mollusk Venoms / Q000737:chemistry*; D010084:Oxidation-Reduction; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D010456:Peptides, Cyclic / Q000138:chemical synthesis / Q000737:chemistry*; D011487:Protein Conformation
D020916:Conotoxins; D004220:Disulfides; D006146:Guanidines; D008978:Mollusk Venoms; D010455:Peptides; D010456:Peptides, Cyclic; C029960:conotoxin GI; D005978:Glutathione; D019791:Guanidine