Correlation between amelogenin primary and secondary structure with its self-assembly

Objective: To elucidate the correlation between primary and secondary structure with self-assembly of amelogenin protein. Methods: Amelogenin sequences from five different vertebrates were analyzed using bioinformatics tools (Protparam, EXPASY). Recombinant full-length porcine amelogenin (rP172) was used to investigate occurrences of secondary structures by circular dicrhroism (CD) and energetic of self-assembly by isothermal titration calorimetry (ITC), at pH = 7.4 with 5 mM CaCl2. Results: Analysis of the primary structures of full-length amelogenin indicated a modular structure containing alternate hydrophobic and hydrophilic domains (blocks) with charged C-terminal amino acid residues. In the mean hydrophobicity vs. mean net charge plot, amelogenin was positioned at the boundary between the intrinsically disordered (natively unfolded proteins) and folded proteins. Using variable temperature CD experiments, we showed that rP172 has polyproline type II (PPII), which is in equilibrium with unordered structures. The unordered/PPII structure was converted to an ordered beta-sheet structure upon self-assembly. ITC measurements confirmed that the assembly is entropically driven due to hydrophobic effect. Conclusions: PPII conformation in amelogenin plays important role in its self-assembly, which are driven mainly by hydrophobic interactions. While amelogenin intrinsically contains flexible and “unstructured” regions, the protein becomes more structured upon self-assembly. We propose that during enamel mineralization stability in amelogenin structure could be achieved either by self-assembly, co-assembly or by its interaction with the mineral. NIH-NIDCR grants: DE-13414, DE-15332.