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, ameloblastins and amelogenins are proteins present during enamel formation and all have been suggested to play a critical role in enamel development. Amelogenins, a family of proteins consisting of a full-length isoform, splice variants and cleavage products, consists of at least 90% of the protein present during enamel growth. The full-length isoform 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. Protein structure is thought to play a key role in the function of amelogenin and it is known that amelogenin forms into a self-assembled quaternary structure called nanospheres which are thought to be tied to the elongated growth of enamel crystals during development. However, insight into the secondary and tertiary structure of amelogenin in nanospheres or bound to hydroxyapatite (HAP) has eluded researchers. No single technique will fully characterize the protein- protein and protein-crystal interactions controlling enamel formation mechanisms, however, recent advancements in several experimental techniques present a unique opportunity to begin addressing some of these critical questions. Building on our previous work, these studies will utilize a suite of techniques including advanced, multi-dimensional solution and solid state NMR, and in situ atomic force microscopy, along with other physical chemistry methods to study critical outstanding questions in the molecular mechanism of enamel formation. Using solution and solid state NMR, the secondary and tertiary structure and the orientation of full-length amelogenin and two naturally occurring mutants will be determined in the nanosphere and bound to HAP, the most biologically relevant forms. The application of these techniques to allow the investigation of proteins >60 residues represents a major advancement for amelogenin specifically and biomineralization proteins in general. To establish which residues which are important in binding to HAP, a series of amelogenin proteins with site-specific amino acid substitutions (Probes) will be made. Crystal growth and binding properties will be characterized using constant composition kinetics and adsorption isotherms. The secondary, tertiary and quaternary structure of Probes with modified crystal growth and interaction properties will be determined on HAP and in the nanosphere. Correlating the structure and orientation results for the Probes compared to native amelogenin will provide crucial insight into the interfacial mechanisms used by amelogenin for exquisite control of enamel. These molecular level insights will allow the implementation of bioinspired designs for therapeutic 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, and is a material that we are unable to reproduce in the lab. 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, limiting our ability to create therapeutic replacements. 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.
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|Zerfaß, Christian; Buchko, Garry W; Shaw, Wendy J et al. (2017) Secondary structure and dynamics study of the intrinsically disordered silica-mineralizing peptide P5 S3 during silicic acid condensation and silica decondensation. Proteins 85:2111-2126|
|Klein, Ophir D; Duverger, Olivier; Shaw, Wendy et al. (2017) Meeting report: a hard look at the state of enamel research. Int J Oral Sci 9:e3|
|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|
|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 P70?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|
|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 T21?I or P41?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|
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