ENERGETIC AND STRUCTURAL BASIS FOR THE PREFERENTIAL FORMATION OF THE NATIVE DISULFIDE LOOP INVOLVING CYS-65 AND CYS-72 IN SYNTHETIC PEPTIDE-FRAGMENTS DERIVED FROM THE SEQUENCE OF RIBONUCLEASE-A

Two fragments of ribonuclease A (RNase A), peptides [61-74] and [65-72], were obtained by solid-phase peptide synthesis. Both peptides contain the residues cysteine-65 and cysteine-72 which form a disulfide bond in native RNase A. The free-energy difference for the formation of the Cys-65-Cys-72 disulfide in the two peptides was obtained from the equilibrium constants measured in aqueous solution at 25-degrees-C and pH 8.0 and 9.0; the free-energy difference was close to zero under these conditions. NOESY and ROESY experiments were carried out in DMSO and in aqueous solution by NMR spectroscopy to determine the conformation of the peptide corresponding to residues 61-74 of RNase A. These experiments show that short-range interactions help to stabilize the Cys-65-Cys-72 disulfide bond with the formation of a type-II beta-turn involving residues 66-69; this turn is shifted by one residue from the native type-III turn at residues 65-68, as deduced in earlier studies of disulfide-forming equilibria in fragments of RNase A.