Alignment of amelogenin ribbons is dependent on a hydrophilic C-terminus

Objectives: The predominantly hydrophobic protein amelogenin is critical for proper mineralization and ultrastructure development in enamel. A role for its hydrophilic C-terminus has mainly been attributed to interactions with mineral ions. In this study we explored the role of the amphiphilic character of the amelogenin and tested the hypothesis that modifications to the C-terminus would primarily affect the protein’s ability to self-assemble and to self-organize into aligned nanoribbons.
Methods: We compared the supramolecular structures of recombinant amelogenins that lacked the hydrophilic domain to amelogenins with additional charged residues at their C-termini using atomic force microscopy and Thioflavin T assays. We also designed a 19-residue-long peptide comprised of portions of the N- and C-termini (NC-19) of amelogenin and studied the propensity to form aligned ribbons in comparison to the N-terminus peptide (N-14) alone.
Results: Using atomic force microscopy and Thioflavin T assays, we observed that a lack of amphiphilicity in these molecules delays ribbon formation significantly as ribbons without the hydrophilic tail take weeks to develop versus days in the amphiphilic protein. The biggest impact of removal of the charged C-terminus is reflected in a lack of self-alignment of the amelogenin ribbons illustrated by short (100-300 nm long) randomly distributed ribbons. Adding charges at the C-terminus of amelogenin accelerated fibril formation, self-alignment and the formation of micrometer-sized bundles of ribbons.
Conclusions: We propose that the presence of the hydrophilic C-terminus of amelogenin is critical for the self-organization of the protein into parallel arrays of nanoribbons and into bundles. Amelogenin bundles are ideal templates for oriented crystallization of apatite fibers that will constitute rod and interrod enamel.