The long range goal of this project is to define the relationships between the conformation of salivary molecules and their biological functions. Initial studies will focus on two molecules found in parotid saliva: (A) the tyrosine-rich phosphoprotein, """"""""statherin"""""""" and (B) the proline-rich glycoprotein, """"""""PRG"""""""". The biological function of greatest importance to this study concerning statherin is the regulation of the calcium-phosphate equilibrium between saliva and the tooth's surface. Regarding PRG, the biologically relevant functions to this project are bacterial clearance from the oral cavity and masticatory lubrication. The empirically-based methods to be used for structural analyses are nuclear magnetic resonance (NMR), circular dichroism (CD), ultraviolet/visible (UV/VIS) and fluorescence spectroscopies. The theoretically-based procedures for elucidation of conformation will include both evaluation of the quantitative results produced through energy minimization and visual inspection of molecular structures generated by computer modeling. The application of these particular methodologies to the aforementioned salivary molecules has already provided detailed information concerning the conformation of the N- glycosylation sites of PRG and promises to further elucidate the three-dimensional structures of native statherin and other functional domains in PRG. Briefly, the procedure for these structural analyses involves first examining the optical (e.g. metal ions, salt, pH, temperature, chaotropic agents, chemical probes, other macromolecules, etc). This information provides data on the bulk secondary and tertiary structures of the proteins/peptides. NMR spectroscopy is then used to elucidate the conformation at the atomic level with further refinements derived from the computer modeling programs. Selected fragments of the native molecules are then tested for biological activity and structurally analyzed. In this way the minimum functional domain still retaining the biological activities of the parent molecule can be isolated. Once this is accomplished, analogs are prepared and tested for enhanced biological activities in the in vitro assay systems. One such proposed functional domain currently being investigated is a cyclic neoglycopeptide analog of the N-glycosylation sites of PRG. Ultimately, these analogs will be used as an aid for the clearance of pathogenic bacteria from the oral cavity and in synthetic salivas for patients with salivary dysfunction.
