Local interactions favor the native 8-residue disulfide loop in the oxidation of a fragment corresponding to the sequence Ser-50-Met-79 derived from bovine pancreatic ribonuclease A.
A 30-residue peptide was obtained from ribonuclease A by chemical cleavage with cyanogen bromide, subsequent sulfitolysis with concomitant S-sulfonation, and finally enzymatic cleavage with Staphylococcus aureus protease. The peptide was converted to the free thiol form by reductive cleavage of the S-sulfo-protecting groups with D,L-dithiothreitol. This peptide consisted of residues 50-79 of the native sequence of ribonuclease A, with the exception that methionine-79 had been converted to homoserine. Included in this sequence are residues cysteine-65 and cysteine-72, which form a disulfide bond in the native enzyme, as well as cysteine-58. This molecule may form one of three possible intramolecular disulfide bonds upon thiol oxidation, viz. one loop of 15 and 2 of 8 residues each. These isomeric peptides were prepared by oxidation with cystamine, 2-aminoethanethiolation of residual thiols, and fractionation by reverse-phase high-performance liquid chromatography. Disulfide pairings were established by mapping the tryptic fragments and confirming their composition by amino acid analysis. After protracted incubation under oxidizing conditions at 25.0 degrees C and pH 8.0, the 26-member ring incorporating the native disulfide bond between residues 65 and 72 is the dominant product. Assuming that equilibrium is established, we infer that local interactions in the sequence of ribonuclease A significantly stabilize the native 8-residue disulfide loop with respect to the non-native 8-residue loop (delta G degree = -1.1 +/- 0.1 kcal mole-1). The implications of this observation for the oxidative folding of the intact protein are discussed.
Milburn
P J
PJ
Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301.
Scheraga
H A
HA
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000941:Antigens / Q000302:isolation & purification; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides; D005136:Eye Proteins / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D012259:Ribonuclease, Pancreatic
D000596:Amino Acids; D000941:Antigens; D004220:Disulfides; D005136:Eye Proteins; D010446:Peptide Fragments; C052615:peptide M, retinal S antigen; D012259:Ribonuclease, Pancreatic