This proposal addresses fundamental questions regarding the processes of protein folding, dimerization and targeting of the protein neurophysin (NP) and its relationship to the disease diabetes insipidus. Neurophysins are small, disulfide-rich proteins that serve as carriers for the hormones oxytocin and vasopressin within the hypothalamo-pituitary tract and that share common precursors with the hormones. Neurogenic diabetes insipidus, which is characterized by inadequate vasopressin secretion, most typically arises from mutations in neurophysin that interfere with its folding, hormone-binding and/or self-association, reflecting the importance of neurophysin to the proper targeting of the hormones to regulated neurosecretor granules. Studies of neurophysin are proposed with the long term objective of elucidating, at the molecular level, the mechanisms by which it folds and by which its dimerization is controlled, and at providing a better understanding of its role in the regulated secretory pathway. These studies will take advantage of recently developed methods for the over-expression of NP in a bacterial expression system. Three areas of research, utilizing spectroscopic, chemical and biological techniques, are specified. 1) The mechanisms by which neurophysin dimerization is modulated by allosteric effects or changes in primary structure will be investigated. Particular emphasis is placed on elucidation of the long range conformation changes that underlie the thermodynamic linkage between peptide-binding and dimerization and on mechanisms by which changes in primary structure, either at or distant from the interface, alter dimerization. 2) Neurophysins are rich in b-structure and contain two homologous domains. Studies are proposed to probe the degree of independence of the two domains during folding, to identify regions of the protein likely to serve as nuclei for folding, and to analyze factors determinant of disulfide pairing and b-structure formation. 3) Mechanisms by which mutations in NP lead to diabetes insipidus will be investigated by correlation of the physical chemical properties of mutant neurophysins with the effects of mutations on the cellula targeting of NP precursors.
| Sardana, V; Carlson, J D; Breslow, E et al. (1987) Chemical modification and cross-linking of neurophysin tyrosine-49. Biochemistry 26:995-1003 |
