The long range goal of this project is to define the relationships between the conformation of salivary macromolecules and their biological functions. Ultimately, this information will be used to develop synthetic saliva for use in patients with salivary dysfunctions. Initial studies will focus on the following two parotid salivary macromolecules: (1) """"""""statherin"""""""" and (2) the proline-rich glycoprotein, """"""""PRG"""""""". The biological functions of statherin and PRG include the regulation of the calcium-phosphate equilibrium between saliva and the tooth's surface, while PRG also takes part in masticatory lubrication, the formation of acquired dental enamel pellicle and bacterial clearance from the oral cavity. The methods of study will be circular dichroism (CD) and 2-dimensional H1-nuclear magnetic resonance (n.m.r.) spectroscopies. This particular combination of methodologies and salivary macromolecules are well suited to each other since: (1) primary sequence data exists for these molecules and (2) they are small enough to undergo rigorous conformational analyses. The specific methods of study will first utilize defined synthetic polypeptide analogs of secondary structural domains as predicted by semi-empirical calculations. Once the presence of that secondary structure is established with CD spectroscopy, the n.m.r. analysis will begin. Using n.m.r. methods, roughly half of the peptide bond angles will be obtained. The remaining peptide bond angles will be specified by computer analysis. This procedure will be repeated for each secondary structural domain until the entire secondary conformation has been mapped out. The next step for statherin will be to proceed from the synthetic polypeptide models to the native, intact molecule. Statherin will be examined by n.m.r. spectroscopy and the tertiary structure specified. Knowing the 3-dimensional architecture of statherin and selected calcium-binding functional domains in PRG, calcium titrations will then be performed and changes in conformation will be elucidated. Based on these results, computer analysis will be used to predict if more effective functional domains can be developed. The improved synthetic analogs of the calcium-binding and/or lubricatory functional domains for statherin and PRG, respectively, will then be prepared and tested for enhanced biological activity.
