Amelogenin: Solution- and solid-state studies of a natural biomineralization process
EMSL Project ID
48235
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 and LRAP, the latter a splice variant of amelogenin that contains only the charged N- and C- termini, are the focus of our four-year, NIH funded investigations. 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 structural studies on full-length amelogen and LRAP 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 LRAP bound to hydroxyapatite. The AFM studies will involve two naturally occurring mutant amelogenin proteins observed in amelogenesis imperfecta. The solution-state NMR structural studies will focus on a Val, Leu, and Ile (?1) methyl-protonated 15N-, 13C-, 2H-labeled amelogenin sample. 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 focus on LRAP because its smaller size allows easy incorporation of selective isotopic labels which 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
2013-12-20
End Date
2014-09-30
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
G.W. Buchko and W.J. Shaw (2015) Improved protocol to purify untagged amelogenin - Application to murine amelogenin containing the equivalent P70&8594T point mutation observed in human amelogenesis imperfecta. Protein Expr. Purif.. 105:14-22.
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, GW Buchko, WJ Shaw, J De Yoreo, and BJ Tarasevich. 2015. "Sequence-defined Energetic Shifts Control the Disassembly Kinetics and Microstructure of Amelogenin Adsorbed onto Hydroxyapatite (100)." Langmuir 31(38):10451-10460. doi:10.1021/acs.langmuir.5b02549
Tarasevich BJ, JS Philo, NK Maluf, S Krueger, GW Buchko, G Lin, and WJ Shaw. 2015. "The Leucine-Rich Amelogenin Protein (LRAP) is primarily monomeric and unstructured in physiological solution." Journal of Structural Biology 190(1):81-91. doi:10.1016/j.jsb.2014.10.007
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