Ribonuclease A: carbon-13 nuclear magnetic resonance assignments, binding sites, and conformational flexibility.

Assignments have been made for 11 methyl, one Gln-C gamma, one Thr-C beta, and all six Tyr-C zeta carbon resonances of ribonuclease A. These partially serve to delineate the binding sites for Cu2+, Mn2+, phosphate, cytidine and its 2'-, 3'-, and 5'-phosphates (Cyd and Cyd-2'-P, -3'-P, and -5'-P), and one or a few urea molecules at low concentration. Evidence is presented for a conformational change, and hence flexibility, in the active site region around the optimum pD for enzymic activity and another such change at around the optimum temperature. The binding of cytidine-containing ligands is shown to have extensive conformational consequences for methyl groups but less for hydrophobic aromatic residues, implying that the former make a special contribution to molecular flexibility. The cytosine ring in Cyd-2'-P, -3'-P, and -5'-P is found to be close but far from parallel to the ring of Phe-120. In contrast to previous claims, ribonuclease A is shown not to unfold even partially before denaturation. On denaturation, it passes to a new but structured state.