pH-Triggered Amelogenin Self-Assembly and the Formation of Higher-Order Structures

Self-assembly of the major enamel matrix protein, amelogenin, is believed to play an essential role in enamel mineralization by regulating the remarkable organization of enamel mineral crystals. Objective: The present study examines the effect of temperature and pH on the self-assembly of amelogenin under near physiological pH conditions. Methods: The effect of pH on protein assembly was explored using dynamic light scattering (DLS), turbidity measurements (595 nm), and TEM analyses. Full-length recombinant amelogenins from mouse (rM179) and pig (rP172) were investigated, along with key degradation products (rM166 and native P148) lacking the hydrophilic C-terminus of the parent molecules. Protein samples (2mg/mL) were prepared in 80 mM TRIS/HCl buffer on ice to study the effect of pH on assembly from pH 8.0 to pH 6.8 at varying temperatures, and at pH 7.2 at 37oC. Results: Results obtained clearly indicate that amelogenin self-assembly is triggered by pH and not by temperature, as frequently reported. Based on DLS findings, very large assemblies formed from particles of similar size (13.5 ± 0.6 nm) for all proteins studied. However, assembly took place at different pH values for each protein studied: pH 7.8 (P148), pH 7.5 (rM166), pH 7.2 (rP172), and pH 7.1 (rM179). Structural differences between the formed assembly products were assessed by turbidity experiments and TEM. The full-length molecules (rM179, rP172) formed tightly connected elongated assemblies of small spheres with high aspect ratio (8 ± 2.0 nm diameter, L/W 7.8), while the truncated molecules formed larger spherical particles that appeared to be loosely associated. Conclusion: The full-length amelogenins form higher order structures different from the truncated amelogenins, suggesting that the hydrophilic C-terminus plays an essential role in the assembly process. Our results suggest that pH triggered assembly can take place under pH conditions reported for secretory enamel. Supported by NIDCR grant DE016376.