Cooperative disulfide bond formation in apamin.
Apamin is being studied as a model for the folding mechanism of proteins whose structures are stabilized by disulfide bonds. Apamin consists of 18 amino acid residues and forms a stable structure consisting of a C-terminal alpha-helix and two reverse turns. This structure is stabilized by two disulfide bonds connecting Cys-1 to Cys-11 and Cys-3 to Cys-15. We used glutathione and dithiothreitol as reference thiols to measure the stabilities of the two disulfide bonds as a function of urea concentration and temperature in order to understand what contributes to the stability of the native structure. The results demonstrate modest contributions from secondary structure to the overall stability of the two disulfide bonds. The equilibrium constants for disulfide bond formation between the fully reduced peptide and the native structure with two disulfide bonds at 25 degrees C and pH 7.0 are 0.42 M2 using glutathione and 2.7 x 10(-5) using dithiothreitol. The equilibrium constant decreases by a factor of approximately 4 in 8 M urea and decreases by a factor of 3 between 0 and 60 degrees C. At least three one-disulfide intermediates are found at low concentrations in the equilibrium mixture. Using glutathione, the equilibrium constants for forming the one-disulfide intermediates with respect to the reduced peptide are approximately 0.025 M. The second disulfide bond forms with an equilibrium constant of approximately 17 M. Thus, apamin folding is very cooperative, but the native structure is only modestly stabilized by urea- or temperature-denaturable secondary structure.
Chau
M H
MH
Department of Biochemistry, Louisiana State University, Baton Rouge 70803-1806.
Nelson
J W
JW
D000595:Amino Acid Sequence; D001030:Apamin / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D004229:Dithiothreitol / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D013329:Structure-Activity Relationship; D013696:Temperature
D004220:Disulfides; D001030:Apamin; D005978:Glutathione; D004229:Dithiothreitol