Enamel biomineralization: A multi-disciplined approach to understand a natural protein-mineral interface. Year 2
EMSL Project ID
49667
Abstract
The protein amelogenin is the central protein controlling the formation of dental enamel, the hardest tissue in the human body. The protein orchestrates the nucleation, growth, and organization of enamel formation (amelogenesis) through the formation of large quaternary structures called nanospheres, consisting of 20-100 monomers. The structure and function of full-length amelogenin is the focus of our five-year, NIH funded investigations. FY17 representes the second year of our project. We request EMSL resources to accomplish three major tasks. (1) In situ-AFM and related studies will allow us to physically view the oligomeric state of amelogenin bound to hydroxyapatite (HAP) surfaces. (2) Solution-state NMR studies on full-length amelogen will enable us to characterize the structure of these proteins under the variety of conditions found during enamel growth. (3) Solid-state NMR studies will enable the quantitative structural characterization of amelogenin bound to hydroxyapatite. The AFM studies will involve a number of “Mechanistic Probes”, protein constructs where residues predicted to be essential for enamel formation have been removed. The solution-state NMR structural studies will focus on a Val, Leu, and Ile (?1) methyl-protonated 15N-, 13C-, 2H-labeled amelogenin samples. Such a labeling scheme in combination with transverse relaxation-optimized spectroscopy (TROSY) experiments, will allow us to track the structural behavior of amelogenin as it self-assembles into large molecular weight complexes. The solid-state NMR studies will be conducted on fully labeled samples and samples with the incorporation of selective isotopic labels to enable the determination of site specific, molecular level protein structure, protein-surface interactions, and dynamics. Structural changes observed in solution or in the solid-state will then be correlated to differences in function.
Project Details
Start Date
2016-11-01
End Date
2017-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Buchko G.W., R.M. Jayasinha Arachchige, J. Tao, B.J. Tarasevich, and W.J. Shaw. 2018. "Identification of major metalloproteinase-20 proteolytic processing products of murine amelogenin and tyrosine-rich amleogenin peptide using a nuclear magnetic resonance spectroscopy based method." Archives of Oral Biology 93, no. 1:187-194. PNNL-SA-132678. doi:10.1016/j.archoralbio.2018.06.001
Jayasinha Arachchige R.M., S.D. Burton, J. Lu, B. Ginovska, L.K. Harding, M.E. Taylor, and J. Tao, et al. 2018. "Solid-State NMR Identification of Intermolecular Interactions in Amelogenin Bound to Hydroxyapatite." Biophysical Journal 115, no. 9:1666-1672. PNNL-SA-131727. doi:10.1016/j.bpj.2018.08.027
Tao J., Y. Shin, R.M. Jayasinha Arachchige, G.W. Buchko, S.D. Burton, A. Dohnalkova, and Z. Wang, et al. 2019. "The energetic basis for hydroxyapatite mineralization by amelogenin variants provides insights into the origin of amelogenesis imperfecta." Proceedings of the National Academy of Sciences of the United States of America 116, no. 28:13867-13872. PNNL-SA-143391. doi:10.1073/pnas.1815654116
Zerfab C, GW Buchko, WJ Shaw, S Hobe, and H Paulsen. 2017. "Secondary structure and dynamics study of the intrinsically disordered silica-mineralizing peptide P5S3 during silicic acid condensation and silica decondensation." Proteins. Structure, Function, and Bioinformatics 85(11):2111-2126. doi:10.1002/prot.25366