AMINO-ACID-SEQUENCE AND PROPERTIES OF VASOPRESSIN-ASSOCIATED ELEPHANTNEUROPHYSIN

The primary structure of an elephant neurophysin, homologous to vasopr essin-associated neurophysins, is reported. The protein contains a Tyr for Asn substitution at position 75, a position in direct contact wit h residues 77 and 78 of the monomer-monomer interface. This Tyr residu e therefore serves as a potential reporter of the path involved in the long-range linkage between peptide binding and dimerization in this s ystem. NMR studies of the protein in unliganded and liganded states de monstrated normal dimerization properties and the expected increase in dimerization associated with binding peptide. In keeping with an elev ated pK(a) of 11.1 assigned to Tyr-75 by UV spectrophotometric titrati on, the NMR signals from the 3,5 and 2,6 ring protons of Tyr-75 were s hifted 0.3 and 0.2 ppm upfield, respectively, relative to their positi ons in small peptides, indicating significant shielding and/or hydroge n bonding. The Tyr-75 ring proton signals narrowed slightly, with no d iscernible change in chemical shift, on conversion from dimer to monom er in the unliganded slate. Ring protons of Tyr-49, distant from the m onomer-monomer interface, but adjacent to the peptide-binding site, we re markedly perturbed by dimerization, in accord with their behavior i n bovine neurophysins. The results suggest that the secondary and tert iary structure of the region 75-78 is largely unchanged by dimerizatio n, and argue against an important role for this region in dimerization -mediated conformational changes that alter the binding site in the un liganded state. On the other hand, binding of peptide was associated w ith both measurable shifts and broadening of Tyr-75 ring NMR signals, consistent with high-pressure studies which have suggested ligand-indu ced changes in the physical properties of the monomer-monomer interfac e. (C) Munksgaard 1994.