title: Dissecting amelogenin protein nanospheres: characterization of metastable oligomers.
abstract: Amelogenin self-assembles to form an extracellular protein matrix, which serves as a template for the continuously growing enamel apatite crystals. To gain further insight into the molecular mechanism of amelogenin nanosphere formation, we manipulated the interactions between amelogenin monomers by altering pH, temperature, and protein concentration to create isolated metastable amelogenin oligomers. Recombinant porcine amelogenins (rP172 and rP148) and three different mutants containing only a single tryptophan (Trp(161), Trp(45), and Trp(25)) were used. Dynamic light scattering and fluorescence studies demonstrated that oligomers were metastable and in constant equilibrium with monomers. Stable oligomers with an average hydrodynamic radius (R(H)) of 7.5 nm were observed at pH 5.5 between 4 and 10 mg · ml(-1). We did not find any evidence of a significant increase in folding upon self-association of the monomers into oligomers, indicating that they are disordered. Fluorescence experiments with single tryptophan amelogenins revealed that upon oligomerization the C terminus of amelogenin (around residue Trp(161)) is exposed at the surface of the oligomers, whereas the N-terminal region around Trp(25) and Trp(45) is involved in protein-protein interaction. The truncated rP148 formed similar but smaller oligomers, suggesting that the C terminus is not critical for amelogenin oligomerization. We propose a model for nanosphere formation via oligomers, and we predict that nanospheres will break up to form oligomers in mildly acidic environments via histidine protonation. We further suggest that oligomeric structures might be functional components during maturation of enamel apatite.
authors.affiliation: Center for Craniofacial Molecular Biology, Ostrow School of Dentistry, University of Southern California, Los Angeles, California 90033, USA.
authors.forename: Keith M
authors.initials: KM
authors.lastname: Bromley
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Kiss
authors.forename: Sowmya Bekshe
authors.initials: SB
authors.lastname: Lokappa
authors.forename: Rajamani
authors.initials: R
authors.lastname: Lakshminarayanan
authors.forename: Daming
authors.initials: D
authors.lastname: Fan
authors.forename: Moise
authors.initials: M
authors.lastname: Ndao
authors.forename: John Spencer
authors.initials: JS
authors.lastname: Evans
authors.forename: Janet
authors.initials: J
authors.lastname: Moradian-Oldak
mesh_terms: D053523:Amelogenin / Q000737:chemistry*; D000818:Animals; D016880:Anisotropy; D005109:Extracellular Matrix / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008027:Light; D054118:Nanospheres / Q000737:chemistry*; D036103:Nanotechnology / Q000379:methods*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence / Q000379:methods; D013552:Swine; D014364:Tryptophan / Q000737:chemistry
chemical_list: D053523:Amelogenin; D011994:Recombinant Proteins; D014364:Tryptophan