title: Quantitatively and kinetically identifying binding motifs of amelogenin proteins to mineral crystals through biochemical and spectroscopic assays.
abstract: Tooth enamel is the hardest tissue in vertebrate animals. Consisting of millions of carbonated hydroxyapatite crystals, this highly mineralized tissue develops from a protein matrix in which amelogenin is the predominant component. The enamel matrix proteins are eventually and completely degraded and removed by proteinases to form mineral-enriched tooth enamel. Identification of the apatite-binding motifs in amelogenin is critical for understanding the amelogenin-crystal interactions and amelogenin-proteinases interactions during tooth enamel biomineralization. A stepwise strategy is introduced to kinetically and quantitatively identify the crystal-binding motifs in amelogenin, including a peptide screening assay, a competitive adsorption assay, and a kinetic-binding assay using amelogenin and gene-engineered amelogenin mutants. A modified enzyme-linked immunosorbent assay on crystal surfaces is also applied to compare binding amounts of amelogenin and its mutants on different planes of apatite crystals. We describe the detailed protocols for these assays and provide the considerations for these experiments in this chapter.
authors.affiliation: Department of Orofacial Sciences, University of California, San Francisco, California, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Zhu
authors.forename: Peter
authors.initials: P
authors.lastname: Hwang
authors.forename: H Ewa
authors.initials: HE
authors.lastname: Witkowska
authors.forename: Haichuan
authors.initials: H
authors.lastname: Liu
authors.forename: Wu
authors.initials: W
authors.lastname: Li
mesh_terms: D000327:Adsorption; D053523:Amelogenin / Q000737:chemistry*; D020816:Amino Acid Motifs; D000818:Animals; D001667:Binding, Competitive; D003460:Crystallization; D003743:Dental Enamel / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006882:Hydroxyapatites / Q000737:chemistry*; D007700:Kinetics; D014074:Tooth Calcification
chemical_list: D053523:Amelogenin; D006882:Hydroxyapatites
keywords: Amelogenin; Biomineralization; Fluorapatite; Hydroxyapatite; Protein–mineral interactions