THE BEHAVIOR OF THE ACTIVE-SITE SALT BRIDGE OF BOVINE NEUROPHYSINS ASMONITORED BY N-15 NMR-SPECTROSCOPY AND CHEMICAL SUBSTITUTION - RELATIONSHIP TO BIOCHEMICAL-PROPERTIES

The active site of liganded neurophysin contains a salt bridge that in volves the side chains of Arg-8 and Glu-47 of the protein and the alph a-amino group of bound hormone or related peptide. The extent to which the Arg-8-Glu-47 salt bridge persists in the absence of peptide, or t o which the environment of Arg-8 in the unliganded state differs in mo nomers and dimers, is relevant to an understanding of allosteric mecha nism in this system. In the present study, the behavior of the salt br idge was investigated by N-15 NMR and chemical replacement of Arg-8. B ovine neurophysin-I was converted to its des 1-8 derivative, and Arg-8 was replaced by N-15-substituted Arg or by other residues using chemi cal semisynthesis. The relative abilities of different amino acids to restore peptide affinity to the des 1-8 protein were in good accord wi th the view of the salt bridge in the liganded state obtained from cry stals of bovine neurophysin-II complexes. In the unliganded state, com parison of the N-15 and proton NMR signals from Arg-8 with those in sm aller arginine systems suggested the absence of significant interactio ns between the guanidinium of Arg-8 and Glu-47 or between the amino te rminal region of Arg-X and other elements of the protein. No evidence of a difference in Arg-8 environment between unliganded monomers and d imers was found. Marked spectral changes accompanying the binding of o xytocin indicated changes in the environment of both the side chain an d amino terminal region of Arg-8. The NMR results were in good agreeme nt with a recently emerging comparison of bovine neurophysin-II deriva tives in the liganded and unliganded states, with the notable exceptio n of the extent of salt bridge formation in the unliganded state. The results are shown to be consistent with, and to help explain, signific ant differences between the two bovine neurophysins in the susceptibil ity to tryptic cleavage at Arg-8 in the unliganded state and in the pH dependence of peptide binding and additionally constrain potential al losteric mechanisms underlying neurophysin ligand-facilitated dimeriza tion.