Dissecting amelogenin protein nanospheres: Characterization of metastable oligomers

Keith M. Bromley, Andrew S. Kiss, Sowmya Bekshe Lokappa, Rajamani Lakshminarayanan, Daming Fan, Moise Ndao, John Spencer Evans, Janet Moradian-Oldak

Research output: Contribution to journalArticlepeer-review

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.Wedid 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 45is 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.

Original languageEnglish (US)
Pages (from-to)34643-34653
Number of pages11
JournalJournal of Biological Chemistry
Volume286
Issue number40
DOIs
StatePublished - Oct 7 2011

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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