Amelogenin proteins constitute the structural framework of the enamel matrix that guides apatite crystal morphology and texture. Self-assembly of amelogenin is required to obtain a three-dimensional scaffold that is able to control mineralization and generate the unique micro- and nanostructures of dental enamel. Amelogenin is known to assemble into nanospheres, but formation of anisotropic and elongated structures of this bipolar molecule seems feasible. Objective: The purpose of this study was to explore if full-length amelogenin protein can assemble into anisometric structures using a variety of physical-chemical and biochemical conditions. Method: Recombinant human full-length amelogenin, rH175, was expressed by E.coli, purified using C4-beads and freeze-dried. Protein was suspended in acid at concentrations between 2 to 8 mg/ml. The pH was adjusted to values between 3.0 and 8.5. Calcium and phosphate were added to selected solutions. 30 µl of protein-solution were placed on glass slides. Microstructure of the gel-matrix was observed using optical, atomic force and electron microscopy. Raman spectroscopy was used to study changes in the secondary structure during self-assembly. Results: Increase in pH above 4 resulted in precipitation of the protein. Fibril formation was observed in a pH range between 4 and 8.5. The fibrils were 5 to 10 µm in diameter and showed birefringence. Fibril diameter was significantly reduced to about 50 nm if calcium and phosphate were added. Atomic force and electron microscopy studies revealed that nanofibers were composed of nanospheres that were aligned in strings. Raman spectroscopy showed a strong shift of amide-I bands to higher wavenumbers (1690 cm-1) during gel-formation of amelogenin, indicating an increase in ß-sheet structure. Conclusions: Amelogenin undergoes hierarchical assembly from nanospheres into nanofibers at high protein concentrations and specific physical-chemical conditions. Support: NIH/NIDCR R21-DE015416 and RO1-DE015821.

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