Laser Raman spectroscopy of biomolecules: 15—Conformational study of partially reduced, fully reduced and cyanogen bromide treated ribonuclease a

Abstract

Reduction of one disulfide bond in bovine pancreatic ribonuclease A, which leaves the enzyme with nearly unimpaired biological activity, makes barely perceptible changes in the aqueous Raman spectrum. Reduction of all four disulfide bonds, which inactivates the enzyme, produces serious conformational changes. The spectrum shows a substantial decrease in a-helical content and conversion of all tyrosines to a weakly hydrogen-bonded form. Cleavage of the peptide chain by cyanogen bromide at the three sites of methionine residues (13, 29-30 and 79) makes little alteration in the a-helical content but reduces the proportion of the P-pleated sheet. This structural change is attributed mainly to the chain breaking at residue 79, which occurs in the center of the P-network.

Local structure involving histidine-12 in reduced S-sulfonated ribonuclease A detected by proton NMR spectroscopy under folding conditions.

“…Although disulfide bonds do not represent the only long-range interactions in a protein, they generally make a major contribution to the stability of the native protein conformation. Several CD (33)(34)(35) and Raman (35,36) spectroscopic studies indicate that significant amounts of local structure are present in reduced RNase A and in reduced cysteine Sblocked derivatives. Immunological studies (37) (38), desalted on Amberlite MB-1 anion/cation exchange resin, and lyophilized from 0.1 M acetic acid at a concentration of <10 mg/ml.…”