STRUCTURAL BIOLOGY OF THE ENAMEL PROTEINS

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Biomineralized tissues are bioceramic-biopolymer composites produced by cell-mediated processes. Current concepts of matrix-mediated biomineralization postulate specific molecular associations between the organic matrix constitutes and the developing inorganic mineral phase such that mineral nucleation and the subsequent control of crystal growth and habit occur in a manner which generates ordered mineralized structures such as bone, dentine and enamel. The determination of the size, shape and location of unique structural motifs of the enamel proteins is an essential prerequisite to advance studies of the functional role of enamel gene products in genetic diseases and in normal enamel biomineralization. Moreover, advances in protein engineering may be expected to lead to the production of new bioceramic materials, perhaps based on principles of enamel formation, with applications in clinical dentistry. Recent studies from this laboratory have established that in the mouse, seven amelogenin proteins are expressed by alternative-splicing from the single X-linked gene and have enabled us to express some of these proteins in a prokaryotic system. We postulate that these amelogenin proteins ar pivotal to the normal formation and development of dental enamel. This Proposal seeks to determine the secondary, tertiary and quaternary structures of the amelogenin proteins. We propose to employ recombinant mouse amelogenins, expressed in both prokaryotic and eukaryotic systems, to characterize and analyze these protein structures. In summary, we propose four Specific Aims: (i) To express, isolate and purify each of the alternatively-spliced murine amelogenins and tuftelins. (ii) To characterize the expressed proteins in terms of their primary structures and post-translational modifications. (iii) To determine the secondary and tertiary structures of amelogenis and tuftelins. (iv) To characterize the quaternary structures of amelogenins and tuftelins; aggregate formation and interactions. Recombinant proteins are isolated from bacterial and/or yeast cell cultures and purified by chromatographic procedures. The primary structures of the expressed proteins are established by amino acid composition and sequence analyses together with mass spectrographic confirmation of molecular weights. The purified characterized proteins are subjected to secondary and tertiary analyses through nuclear magnetic resonance (NMR), circular dichroism (CD) and protein crystallization - X-ray diffraction procedures. Protein-protein interactions and aggregate self-assembly mechanisms are characterized by a combination of dynamic light scattering (DLS), high-resolution transmission electron microscopy (TEM), size-exclusion chromatography (SEC) and atomic force microscopy (AFM). Finally, data derived from these studies are collated to provide molecular models for amelogenin structures and for the putative specific protein-mineral and protein-protein interactions required for the regulation of dental enamel biomineralization.