Acidic proteins found in mineralized tissue act as biology's crystal engineers. Their activities are responsible for the material properties of hard tissues, and their activities directly control the hierarchical architecture of these tissues. However, despite their importance in such fundamental physiological processes as bone and tooth formation, there is remarkably little known of the protein structure-function relationships which govern crystal recognition. The primary goal of this program is to obtain a molecular description of the structure-function relationships used by small acidic proteins in the crystal engineering of hydroxyapatite and calcium oxalate (the principle mineral phases of bone/teeth and kidney stones, respectively). Preliminary genetic engineering results with a small model protein, consisting structurally of a long alpha-helix and two antiparallel beta-sheets, have demonstrated that the protein surface electrostatic charge distribution can directly dictate whether secondary nucleation and crystal growth is promoted or inhibited. The molecular recognition mechanisms underlying this observation will be studied with a combination of kinetic and thermodynamic techniques, along with direct solid-state NMR determination of the protein-crystal interfacial structure. Further studies will characterize how secondary structure scaffolds are used to present carboxylate side-chains with stereospecificities that direct interactions with hydroxyapatite and calcium oxalate. Genetic engineering techniques will be used to systematically place carboxylate side-chains on the alpha-helix and anti- parallel beta-sheets, and the subsequent molecular interactions with the crystals will again be studied with solid-state NMR techniques. In addition, the functional properties of these proteins will be assessed using a variety of techniques including constant composition kinetics, direct binding adsorption analysis, particle size determination, electron microscopy, and zeta-potential measurements. The disruption of normal biomineralization processes can lead to pathological mineralization or demineralization, such as in atherosclerotic plaque formation, artificial heart valve calcification, kidney stone build-up, dental calculus formation, or bone and tooth demineralization. A better understanding of the biomolecular mechanisms used to promote or retard crystal growth could provide important design principles for the development of calcification inhibitors and promoters in orthopaedics, cardiology and dentistry.
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