Stabilities of disulfide bond intermediates in the folding of apamin.
Apamin is an 18-residue bee venom peptide with the sequence CNCKAPETALCARRCQQH-amide and contains 2 disulfide bonds connecting C-1 to C-11 and C-3 to C-15. In the folding of reduced, unfolded apamin to native apamin with two disulfide bonds, the one-disulfide folding intermediate states are not populated to significant levels. To study the properties of the one-disulfide intermediates, we have synthesized two peptide models to mimic the one-disulfide intermediates, Apa-1 and Apa-2, in which two cysteines in the sequence have been replaced by alanines. These peptides can form only one of the native disulfide bonds, C-1 to C-11 in the case of Apa-1 and C-3 to C-15 in the case of Apa-2. The stabilities of these disulfide bonds have been measured as a function of pH, concentration of urea, and temperature, in order to understand which contributions stabilize the disulfide-bonded structures. Using oxidized and reduced glutathione, the equilibrium constants for forming the disulfide bonds at 25 degrees C and pH 7.0 are 0.018 M for Apa-1 and 0.033 M for Apa-2 and show little dependence on pH or temperature. Both disulfide bonds are destabilized slightly (by approximately a factor of 2) between 0 and 8 M urea. Circular dichroism spectra indicate that although both Apa-1 and Apa-2 exhibit some structure, Apa-2 exhibits more than Apa-1. The results suggest that in the folding of apamin, the one-disulfide intermediate containing the C-3 to C-15 disulfide bond, as in Apa-2, is favored slightly. Secondary structure provides modest stabilization to this intermediate.
Huyghues-Despointes
B M
BM
Department of Biochemistry, Louisiana State University, Baton Rouge 70803-1806.
Nelson
J W
JW
D000595:Amino Acid Sequence; D001030:Apamin / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011487:Protein Conformation / Q000187:drug effects; D013696:Temperature; D014508:Urea / Q000494:pharmacology
D004220:Disulfides; D001030:Apamin; D014508:Urea