The overall goal of this research is to elucidate the interfacial mechanisms of the biomineralization proteins driving the formation of enamel. Enamel is the most highly ordered biomineralization crystal and is uniquely designed to handle abrasions and mechanical stress. Enamelins, tuftelins, ameloblastins and amelogenins are proteins present during enamel formation and all have been suggested to play a critical role in enamel development. Amelogenin consists of 90% of the protein present during enamel growth, is necessary for proper enamel formation and as such, it is the primary focus of the proposed studies. Very little is understood at a mechanistic level about how amelogenin controls crystal growth. It is known that amelogenin forms into unique self assembled nanospheres which are thought to be tied to the elongated growth of enamel crystals during development. However, the organization of the nanosphere is not well defined, and the protein- hydroxyapatite interface is not understood on a molecular level. Protein structure is thought to play a key role in the function of amelogenin as a possible crystal nucleator and growth regulator, but insight into the secondary and tertiary structure of amelogenin has eluded researchers. No single technique will fully characterize the protein-protein and protein-crystal interactions controlling enamel formation mechansims, however, recent advancements in several experimental techniques present a unique opportunity to begin addressing some of these critical questions. Relating the protein-protein and protein-surface interactions to function will be the emphasis of the proposed work, particularly focusing on the loss of function as a result of mutation. Building on our previous work,these studies will utilize a suite of techniques including solution and solid state NMR, atomic force microscopy (AFM), quartz crystal microbalance (QCM), constant composition kinetics (CCK) and molecular modeling to study critical outstanding questions in the molecular mechanism of enamel formation. Using NMR, the secondary structure and the orientation of naturally occurring mutants will be determined and compared to the structure of the wildtype protein. The affect of pH, ionic strength and protein concentration will also be investigated. AFM will be used to determine the quaternary structure of the adsorbed protein. Protein-protein interactions will be determined using solution state NMR, revealing precise residues involved in nanosphere self-assembly. To provide a correlation between structure and function, QCM and CCK will be used to investigate nucleation rates, growth inhibition and crystal modification under identical conditions used in the structural studies. Correlating the structure and orientation results with differences in growth and nucleation under similar conditions will provide crucial insight into the interfacial mechanisms used by amelogenin for exquisite control of the enamel matrix. These insights are necessary for the design of theraputic solutions to deficient enamel. More generally, these studies will provide basic insight into protein/crystal interactions dominating the formation of all biominerals.
Enamel is the most highly mineralized tissue in the body, and produces hydroxyapatite crystals with a strength approaching that of steel. Amelogenin is a protein that is critical to the formation of this highly organized material, but how it controls enamel formation is not understood on a molecular level. Using a combination of the most advanced techniques available, we propose to elucidate the protein-protein and protein-hydroxyapatite interaction mechanisms, insights that are necessary before long-lasting therapeutics can be designed.
|Tao, Jinhui; Buchko, Garry W; Shaw, Wendy J et al. (2015) Sequence-Defined Energetic Shifts Control the Disassembly Kinetics and Microstructure of Amelogenin Adsorbed onto Hydroxyapatite (100). Langmuir 31:10451-60|
|Buchko, Garry W; Shaw, Wendy J (2015) Improved protocol to purify untagged amelogenin - Application to murine amelogenin containing the equivalent P70â†’T point mutation observed in human amelogenesis imperfecta. Protein Expr Purif 105:14-22|
|Shaw, Wendy J (2015) Solid-state NMR studies of proteins immobilized on inorganic surfaces. Solid State Nucl Magn Reson 70:1-14|
|Tarasevich, Barbara J; Philo, John S; Maluf, Nasib Karl et al. (2015) The leucine-rich amelogenin protein (LRAP) is primarily monomeric and unstructured in physiological solution. J Struct Biol 190:81-91|
|Lu, Jun-Xia; Burton, Sarah D; Xu, Yimin S et al. (2014) The flexible structure of the K24S28 region of Leucine-Rich Amelogenin Protein (LRAP) bound to apatites as a function of surface type, calcium, mutation, and ionic strength. Front Physiol 5:254|
|Buchko, Garry W; Lin, Genyao; Tarasevich, Barbara J et al. (2013) A solution NMR investigation into the impaired self-assembly properties of two murine amelogenins containing the point mutations T21â†’I or P41â†’T. Arch Biochem Biophys 537:217-24|
|Tarasevich, Barbara J; Perez-Salas, Ursula; Masica, David L et al. (2013) Neutron reflectometry studies of the adsorbed structure of the amelogenin, LRAP. J Phys Chem B 117:3098-109|
|Lu, Jun-xia; Xu, Yimin Sharon; Shaw, Wendy J (2013) Phosphorylation and ionic strength alter the LRAP-HAP interface in the N-terminus. Biochemistry 52:2196-205|
|Lu, J X; Xu, Y S; Buchko, G W et al. (2013) Mineral association changes the secondary structure and dynamics of murine amelogenin. J Dent Res 92:1000-4|
|Masica, David L; Gray, Jeffrey J; Shaw, Wendy J (2011) Partial high-resolution structure of phosphorylated and non-phosphorylated leucine-rich amelogenin protein adsorbed to hydroxyapatite. J Phys Chem C Nanomater Interfaces 115:13775-13785|
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