NMR BEHAVIOR OF THE AROMATIC PROTONS OF BOVINE NEUROPHYSIN-I AND ITS PEPTIDE COMPLEXES - IMPLICATIONS FOR SOLUTION STRUCTURE AND FOR FUNCTION

The NMR behavior of the aromatic protons of bovine neurophysin-I and i ts complexes was interpreted with reference to the 2:8 Angstrom crysta l structure of the dipeptide complex of bovine neurophysin-II and to m echanisms underlying the thermodynamic linkage between neurophysin dim erization and peptide binding. Large binding-induced shifts in the rin g proton signals of Tyr-2 of ligand peptides (similar to 0.5 ppm upfie ld and similar to 0.35 ppm downfield at 25 degrees C for the 3,5- and 2,6-ring protons, respectively) were demonstrated. Consistent with the crystal structure, and in disagreement with conclusions by other inve stigators, evidence is presented indicating the absence of dipolar Con tact between Tyr-2 ring protons and protein Phe ring protons. The larg e binding-induced shifts are attributed to a strong influence of proxi mal neurophysin carbonyl and disulfide groups on the bound Tyr-2 ring, of potential importance in binding specificity. Resolution of the beh avior of neurophysin Phe residues -22 and -35 and of their proton NOE contacts provided insights into the conformational changes associated with peptide binding and with dimerization. Within the amino domain of the protein, as evidenced by the behavior of interface residue Phe-35 and its NOE contacts, binding-induced changes in the subunit interfac e appeared to involve principally the junction between this interface region and the 3,10-helix that connects it to the binding site in the liganded state. By contrast, as judged by the NOE contacts of His-80, the corresponding interface participant of the carboxyl domain, peptid e binding induced a marked decrease in side-chain mobility within the carboxyl domain segment of the interface, Interactions of Phe-22 with protons assigned to Ala-68, neither of which is an interface participa nt, were demonstrated to be markedly altered both by dimerization in t he unliganded state and by peptide binding to the dimer. Since Phe-22 is adjacent to the peptide-binding site, the results collectively supp ort a model in which conformational differences between unliganded mon omer and dimer are important contributors to the preferential binding of peptide to the dimer and indicate that the amino and carboxyl domai n segments of the Interface, which are homologous, are affected differ ently by peptide binding.