Calcium ion (Ca²⁺) concentration affects stability of amyloid mediated self-assemblies of amelogenin peptides

Objectives: Tooth enamel is made of highly organized apatite matrix. The biomineralization of apatite during amelogenesis is believed to be modulated by amelogenin. In presence of calcium, recombinant human full-length amelogenin (rH174) and its peptide (14P2) can self-assemble into nanoribbons in vitro. These are believed to guide apatite growth in developing enamel in vivo. Recent studies suggest that 14P2 region within amelogenin strongly interacts with itself enabling the formation of β-sheets similar to those seen in amyloids. However, the specific role of calcium ion on 14P2 self-assembly has not been defined. Here we investigate 14P2 self-assembly at different calcium concentrations using atomic force microscopy (AFM) and Thioflavin-T (ThT) assays.
Methods: Samples of 14P2 at a final concentration of 0.6 mM were prepared in five test tubes containing 0.0, 0.13, 1.3, 2.7 or 13 mM CaCl₂ and KH₂PO₄ at a ratio of 1.6:1. Solutions were then adjusted to pH 5.0. Phosphorylated 14P2 (_p14P2) samples were assembled in similar conditions only without KH₂PO₄. Samples were concentrated ~4 times by evaporation for three hours at 37°C. AFM was used to visualize ribbon density. Effects of calcium concentration on the kinetics of ribbon formation were quantified using ThT assay with fluorescence spectroscopy.
Results: In the presence of calcium ions, 14P2 and _p14P2 assembled into nanoribbons measuring 3.0 (±0.9) and 5.9 (±2.0) nm in height respectively. _p14P2 ribbons had a higher tendency for bundling than 14P2. Both 14P2 and _p14P2 nanoribbons were most abundant in peptide samples assembled in high calcium concentration (13 mM) and were scarce in samples with low (0.13 mM) or no calcium (0 mM) content. ThT fluorescence intensities increase faster with increasing calcium content and therefore suggest improved or accelerated amyloid ribbon assembly. However, readings for 14P2 at various calcium concentrations were not significantly different from one another.
Conclusions: 14P2 and _p14P2 amyloid-mediated self-assembled nanoribbons can develop withiout the addition of of Ca²⁺ ions, but those assemblies appear less stable. The presence of divalent Ca²⁺ may allow bridges to form between amelogenin dimers and thus stabilize the fomation of β-sheet aggregates as suggested in a recent molecular model of the amelogenin amyloid structure.