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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.

The interaction between molecules is essential in a wide range of biological processes. A detailed knowledge of these interactions is necessary for understanding these processes. Among the systems that involve important interactions is the immune system. NMR spectroscopy has a large number of spectral parameters that were used in this work to study antibody-antigen interactions. These same parameters were also used to begin a structural analysis of a medium-sized protein, neurophysin, that has important interactions with neurohormones, and served here as a model antigen. A set of ligands differing in size and charge was designed and used to probe the binding site of anti-phosphocholine antibodies. These ligands ranged from small organic species to medium sized proteins. Amino acids, peptides and proteins were homogeneously linked to phenyl phosphocholine and analyzed by NMR techniques. Transferred nuclear Overhauser effect measurements were used to determine the conformation of bound ligands. The conformational change observed in some ligands was explained as either due to the antibody selecting one conformation that already exists, or the antibody binding inducing a conformational change. Titration data and detailed NMR analysis showed a more rigid M3C65 antibody fragment upon binding. In summary, with eight examples of ligands and four examples of antibodies studied by NMR, a spectrum of effects was seen, including a lock-and-key model and limited local induced fit. The contribution of the carrier molecule to antibody binding was in restricting the conformation of the ligand. Bigger ligands that are expected to be more immunogenic, showed less binding avidity as determined by immunological assays. Fluorinated ligands were synthesized to determine the kinetics of binding using 19F NMR spectra. Higher concentration of a fragment of the antibody M3C65 was analyzed to determine assignments of some residues in the combining site of the antibody. High resolution NMR techniques were used to assign resonances in neurophysin. The physiological role of neurophysin includes hormone storage and stabilization of oxytocin and vasopressin against proteolytic degradation within the posterior pituitary. Neurophysin is a 10 KD protein that dimerizes at high concentrations needed for NMR studies. An organic cosolvent was used to lower the dimerization constant, and hence inrease the spectral resolution. This permitted sequence-specific assignments that were then used to identify residues in the neurophysin-hormone binding site. Chemical shift differences and conformational changes were observed for the residues glutamate 47 and leucine 50. The 3₁₀ helix was further stabilized towards a more ideal helix upon hormone-analog peptide binding. Some of the residues contributing to the monomer-monomer interface were also assigned. Dimerization ill1duced chemical shift differences and conformational changes were observed for phenylalanine 35, threonine 38, and alanine 69. Tyrosine: 49 and phenylalanine 22 were affected but to a lesser extent. One characteristic of neurophysin in all studied cases was dynamic equilibrium between different folding states.