Enamel, the outermost covering of teeth forms extracellularly through the ordered assembly of a protein scaffolding that regulates crystallite formation. The underlying mechanism for protein assembly within the enamel extracellular matrix and the regulatory roles for protein to protein and crystallite to protein interactions are not known. Amelogenesis imperfecta, genetic defects of enamel, include several forma with mutations in the human amelogenin locus that affect amelogenin expression, resulting in alterations in protein stoichiometry and/or the loss of protein domains required to regulate enamel organic matrix biogenesis. An emerging view of enamel biogenesis includes protein to protein interactions essential for assembly of the enamel organic matrix since protein to protein interaction will affect the spatial organization of the matrix that provides protein to crystallite regulatory interactions. THE HYPOTHESIS UNDERLYING THIS APPLICATION IS THAT SUPRAMOLECULAR ASSEMBLY OF THE ENAMEL ORGANIC MATRIX REQUIRES SPECIFIC PROTEIN DOMAINS AND IT IS THIS PROTEIN ASSEMBLY THAT ULTIMATELY DIRECTS BIOMINERALIZATION. We will use the yeast two-hybrid system to identify and define domains essential to protein to protein interactions used during supra molecular assembly of the enamel organic matrix. We will search for protein(s) other than amelogenin and tuftelin that interact with amelogenin and participate during protein assembly of the enamel organic matrix. To test our hypothesis we propose five interrelated specific aims: 1) To use the yeast two hybrid system to screen for specific enamel protein interactions; 2) To identify and map minimal domains required for specific enamel protein interactions; 3) To identify unknown enamel protein(s) associated with enamel organic matrix assembly; 4) To map minimal protein domain(s) required for interactions among newly identified enamel matrix proteins; 5) To perturb assembly functions of the identified domains by site-directed mutagenesis. Successful completion of these aims will permit critical insights into the assembly and disassembly of the enamel extracellular matrix during biomineralization. Such insights can be used to model the molecular basis for enamel defects resulting from environmental insults, such as fluoride, or from genetic disorders, such as in amelogenesis imperfecta. Identification of the minimal protein domains and their structure will provide enhanced understanding of protein structures which are required for enamel matrix self-assembly.
