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

doi:10.1074/jbc.m111.250928

Keith M Bromley, Andrew S Kiss + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m111.250928

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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.

Highlights

Tooth enamel formation follows the general organic matrixmediated biomineralization process in which the extracellular matrix components regulate the nucleation, growth, and orga
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
In various protein systems, such as the amyloid protein A␤40 [10, 11], Syrian hamster prion protein [12], hydrophobin SC3 [13], and tobacco mosaic virus [14], a metastable oligomeric form has been identified as forming the “building blocks” for or being a necessary precursor to the formation of larger organized protein structures

Summary

Introduction

Tooth enamel formation follows the general organic matrixmediated biomineralization process in which the extracellular matrix components regulate the nucleation, growth, and orga-. Amelogenin accounts for ϳ90% of the protein content of the developing enamel matrix, and micrographs have revealed that it may exist in the form of self-assembled nanochains of nanospheres [2]. With regard to secondary structure, amelogenin has been shown recently [4, 15] to belong to a growing class of proteins known as intrinsically disordered proteins (IDPs)3 [16]. This disorder is most prominent when amelogenin is forced into a monomeric state, which can be achieved by dissolving the protein in acidic conditions.

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