IDENTIFICATION AND OBSERVATION OF ALKYL PROTON RESONANCES OF THE AMINO‐TERMINAL RESIDUES OF BOVINE NEUROPHYSINS

Abstract

Analysis of the 220 MHz proton magnetic resonance spectra of bovine neurophysins-I and -II and of the effects of pH and succinylation on these spectra has allowed identification of the -CH₃ proton resonances of the amino-terminal alanine of both proteins and of the -CH₃ resonance of methionine-2 of neurophysin-II. The alanine -CH₃ resonance of neurophysin-I is a sharp doublet at all pH values between 1 and 10.5 indicating relatively few restrictions on its mobility. By contrast, the -CH₃ resonances of the amino-terminal alanine and methionine-2 of neurophysin-II undergo pH-dependent changes in broadening compatible with the formation of an intramolecular salt-bridge at neutral pH between the protonated α-amino and an unprotonated side chain carboxyl. The results suggest that differences in the properties of the two proteins are partially mediated by conformational differences involving their amino-terminal sequences. The potential usefulness of the amino-terminal resonances as n.m.r. ‘reporter’ signals is additionally demonstrated by studies of the effects of spin labels on the neurophysin-I amino-terminal alanine resonance; these studies place the amino-terminus of neurophysin-I approximately 14 Å from residue 3 of peptides bound to the strong neurophysin hormone-binding site.