Self-Assembly of Amelogenin Proteins Modified for Templated Mineralization

Objectives: Amelogenin is the predominant (90%wt) enamel matrix protein. It self-assembles into a supramolecular framework that, combined with other proteins, facilitates the orientation and morphology of apatite enamel crystals. However, the supramolecular matrix and the mechanisms by which such biomolecules control the nucleation, growth, and morphology of mineral is far from understood. Here we tested the hypothesis that extension to the amelogenin protein by amino acid sequences that are known to affect mineralization may facilitate guided-growth of apatite fibers on an organic template.

Methods: Recombinant full-length amelogenin (rH174) was modified by attaching aspartic acids (rH174-Asp), glutamic acid (rH174-Glu), and statherin (rH174-StC,-StN,-StLC) to its N- or C-terminal. Enamelin peptide with calcium and phosphate(Ca²⁺/PO₄^3-) was added to existing rH174 nanoribbons at pH7 in various conditions, while other samples were incubated in various concentrations of Ca²⁺/PO₄^3- in solution and buffers at pH5.5. Samples were prepared and imaged on mica and glass. Self-assembly and nanoribbon dimensions and mineralization were analyzed by atomic force microscopy(AFM) and transmission electron microscopy(TEM).

Results: Nanoribbons did not form for rH174-StLC, while all other modification self-assembled into ribbons within 2 weeks. They exhibited high organization and alignment (rH174-Asp,-Glu,-StN,-StLC), networked aggregation (rH174-Glu), and random orientation (rH174-Asp -StN) under various conditions. rH174-Glu exhibited the most consistent nanoribbon structures. Analysis by TEM indicated the presence of randomly-oriented crystalline mineral in rH174-Asp at 8 weeks.

Conclusions: The addition of nucleating domains to the full-length amelogenin protein appear to not drastically alter the self-assembly behavior of amelogenin. Interaction with delivery agents may allow for guided growth of apatite fibers on these functionalized molecules.