Amelogenins comprise the majority of proteinaceous material in secretory stage enamel and are important in the regulation of enamel crystal growth during mineralization. The overall objective of this proposal is to begin to study the molecular structure of amelogenins by single crystal X-ray crystallographic techniques. The determination of the three dimensional structure of these proteins is necessary to fully understand their role in the interaction with calcium, phosphates fluoride, and other components of developing enamel.
The specific aims of this proposal are: 1) to prepare milligram quantities of the major amelogenins from bovine secretory enamel; 2) to prepare X-ray diffraction-quality single crystals of one or more of the major amelogenins found in bovine enamel and to characterize these crystals by measuring the unit cell dimensions, estimating the quality and extent of diffraction and assignment of space group symmetry; 3) to collect a native data set of one or more of the amelogenins and begin screening experiments to identify heavy atom derivatives. The major amelogenins will be isolated from bovine secretory enamel and purified by isoelectric focusing, gel permeation chromatography, and reverse phase HPLC. Preliminary studies have demonstrated that this procedure will yield enough pure amelogenin to begin a systematic screening procedure necessary to refine the optimal protein crystal growth conditions. The crystals will be examined by standard X-ray diffraction techniques and native data will be collected for the crystal form which demonstrates the highest resolution data. Soaking of similar crystals in solutions of appropriate heavy atoms will be performed until a suitable isomorphous derivative can be identified. These studies are a required prerequisite to the three dimensional structure determination of amelogenin. The visualization of the molecular architecture of amelogenin will improve our understanding of the precise details of the interaction of this molecule with others during the mineralization process. The particular amino acids are important to the structure and thus the function of amelogenin can then be elucidated.
