This proposal is aimed at determination of the structure of neurophysin-hormone complexes, elucidation of the structural basis of the thermodynamic linkage between hormone-binding and neurophysin self-association and calculation of the thermodynamics of bonding between individual segments of peptide and neurophysin. Basic structural studies will include determination of the mode of disulfide pairing in neurophysin and continued proton NMR and fluorescence studies of distance relationships within neurophysin- peptide complexes. The mechanism underlying the preferential binding of hormone to the neurophysin dimer will be probed by proton NMR, circular dichroism and fluorescence studies of the relative conformations of liganded and unliganded neurophysin monomers and dimers and of the spatial relationship between bound hormone and the subunit interface of the dimer. Resonance Raman will be used to investigate the apparent non-equivalence of neurophysin subunits within the dimer. The significance of apparent differences in self-association in the crystal and in solution will also be investigated in this context. The thermodynamics of bonding between individual hormone residues and neurophysin will be further evaluated via additional studies of intermolecular complex formation and via studies of bonding between hormone and neurophysin segments of the semi-synthetic common precursors of hormone and neurophysin. Aspects of the bonding of the hormone tyrosine to neurophysin will additionally be probed by resonance Raman spectroscopy for correlation with thermodynamic analysis. Finally, as part of a collaborative study, efforts will be made to express the gene product of neurophysin cDNA in culture in order to probe specific bonding interactions by site specific mutagenesis, to obtain precursor species not available by semi- synthesis and to prepare isotopically substituted protein for NMR analysis. The role of this laboratory in the project will be to investigate the conformational and functional properties of the protein so produced.
| Sardana, V; Carlson, J D; Breslow, E et al. (1987) Chemical modification and cross-linking of neurophysin tyrosine-49. Biochemistry 26:995-1003 |
