Characterization of Metastable Oligomers as Subunits of Amelogenin Protein Nanospheres

Abstract

Amelogenin protein accounts for more than 90% of the protein content during tooth enamel biomineralization and controls the organized growth of enamel apatite crystals. The protein self-assembles into nanospheres in vitro. We manipulated the interactions between monomers by altering pH, temperature and protein concentration to create isolated metastable oligomers. Recombinant amelogenin (rP172), and three different mutants containing only a single tryptophan (W161,W45,W25) were used. We observed stable but unstructured oligomers with an average hydrodynamic radius (RH) of 7.5 nm at pH 5.5. Fluorescence experiments with single-tryptophan amelogenins revealed that upon oligomerization, the C-terminus is exposed at the surface of the oligomers, while the N-terminal region is involved in self-assembly. Schematics of the monomeric, oligomeric and nanosphere forms of amelogenin are indicated at the relevant pH in the figure; A plot of wavelength emission maxima of amelogenin over a range of pH values between 3.4 and 9.4. The blue-shift in emission wavelength was most apparent at around pH 6. We propose that nanospheres form via oligomers. We predict that nanospheres will break up to form oligomers in mildly acidic environments in vivo and might be functional components during enamel maturation.View Large Image | View Hi-Res Image | Download PowerPoint Slide