Neurophysins (NPs) make up a relatively small, stable, and highly soluble class of proteins. They have physiological roles of storage and stabilizing' of peptide hormones oxytocin and vasopression within the posterior pituitary neurosecretory granules. At the concentration of NP found within the granules, NP would exist as a dimer in the absence or presence of bound peptide. The NP monomer-monomer interface involves B-sheet/ B-sheet contact, which can be modulated by the presence of cosolvent. This remarkable feature of NP makes it a model for Alzheimer's disease. One of the characteristics of Alzheimer's disease is the presence of plaques of B-amyloid protein that are deposited on the brain. The plaques are rich in B-structure. Being water-insoluble makes them impossible to be directly studied by solution-state NMR. The purpose of this study was to modify the solvent system to lower the NP dimerization constant and characterize the nature of solvent on dissociation of dimer. A set of cosolvents was selected to try to reduce NP dimerization at relatively high concentration of NP. The organic cosolvents included deuterated methanol, dimethyl sulfoxide, ethyl acetate, propionitrile, and acetonitrile. Also, the protein unfolding reagents, deuterated urea and guanidine monohydrochloride, were tried. The interaction between bromophenol blue and NP was also studied because this dye binds predominately to the dimer form of NP. Highresolution NMR techniques were used to sense the NP-I dimer I monomer equilibrium. Among the organic cosolvents used, only acetonitrile and propionitrile were found shift the dimer ~ monomer equilibrium significantly toward monomer. The cosolvent probably changed the character of the solvent system, penetrated the monomer-monomer interface and interacted with the interface residues, caused the break up of dimer. The unfolding reagents were found to partly unfold the NP simultaneously with dissociation of the dimer. Bromophenol blue binds to NP-I at low pH, but the solubility of NP-dye complex is too low to be studied extensively by solution-state NMR methods.
| Identifer | oai:union.ndltd.org:pdx.edu/oai:pdxscholar.library.pdx.edu:open_access_etds-6281 |
| Date | 29 April 1996 |
| Creators | Yao, Jian |
| Publisher | PDXScholar |
| Source Sets | Portland State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Dissertations and Theses |
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