Initial disulfide formation steps in the folding of an omega-conotoxin.
To determine whether the native disulfides of omega-conotoxins are preferentially stabilized early in the folding of these small proteins, the rates and equilibria for disulfide formation were measured for three analogues of omega-conotoxin MVIIA. In each analogue, one of the three pairs of disulfide-bonded Cys residues was replaced with Ala residues, leaving four Cys residues that can form six intermediates with one disulfide and three species with two disulfides. For each analogue, all of the disulfide-bonded species were identified, and the equilibrium constants for forming the individual species via exchange with oxidized and reduced glutathione were measured. These equilibrium constants represent effective concentrations of the Cys thiols and ranged from 0.01 to 0.4 M in the fully reduced protein. There was little or no preference for forming the native disulfides, and the equilibria for forming the first and second disulfides decreased only slightly upon the addition of 8 M urea. The data for the four-Cys analogues, together with equilibrium data for the six-Cys form, were also used to estimate effective concentrations for forming a third disulfide once two native disulfides are present. These effective concentrations were approximately 100 and 10 M in the presence of 0 and 8 M urea, respectively. The results indicate that there is little or no preferential formation of native interactions in the folding of these molecules until two disulfides have formed, after which there is a high degree of cooperativity among the native interactions.
Price-Carter
Marian
M
Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112-0840, USA.
Bulaj
Grzegorz
G
Goldenberg
David P
DP
D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D017510:Protein Folding; D020960:omega-Conotoxins / Q000737:chemistry / Q000378:metabolism*
D004220:Disulfides; D020960:omega-Conotoxins; D003545:Cysteine