The Effect of HCl Incubation on the Self-assembly of Recombinant Amelogenin

Objectives: Amelogenin is the major protein present in developing enamel and plays a key role in enamel mineralization. Its self-assembly behavior in different conditions remains an area of debate. Previously, we reported on the self-assembly of amelogenin (rH174) into amyloid-like ribbons in the presence of calcium and phosphate ions. Self-assembly kinetics was inconsistent, with ribbons developing sometimes within days, mostly within a couple of weeks, and occasionally within six months. In this study we explored how changes in pH influences ribbon formation kinetics and tested the hypothesis that dissolution and incubation of lyophilized amelogenin in HCl solution is beneficial to obtain consistent assembly kinetics.
Methods: 1 mg of amelogenin (rH146, rH174, rH174(+9)) was dissolved in 1 mM HCl (pH ~ 2) and incubated for 5 min to 60 minutes. Next, CaCl₂ and KH₂PO₄ solutions were added to the solution, with a final concentration of 33.4 mM and 20.9 mM, respectively. The pH of the sample solution was then adjusted to 3 or 5. Protein concentration was increased by a factor of 4 via solvent evaporation. Atomic force microscopy (AFM) was used to characterize assembly into nanoribbons.
Results: Increasing the time of incubation in 1 mM HCl prior to self-assembly in calcium and phosphate solutions from 5 to 60 minutes significantly improved reproducibility and assembly kinetics of all four constructs. After one week of post-assembly, randomly distributed nanoribbons were observed; and within three weeks ribbons self-aligned and formed bundles that were several micrometers in length.
Conclusions: Our results show that dissolution of freeze-dried amelogenin in HCl solution is a crucial step for reproducibility of amelogenin self-assembly into nanoribbons. Acidity will induce protonation of amino acid residues, which may be required to completely unfold amelogenin molecules and dissociate intermolecular bonds established during freeze-drying. Increased assembly reproducibility may occur due to proper protein dissolution. At the same time, folding allows intermolecular hydrophobic interactions and hydrogen bonding upon pH increase and addition of calcium and phosphate ions.