Physical Chemical Conditions for Hierarchical Amelogenin Self Assembly

Introduction: Amelogenin is the main compound of the enamel matrix and is known to induce anisotropic apatite crystal growth during enamel mineralization. Amelogenin self-assembly leads to the formation of nanospheres. Hierarchical self-assembly of the protein into elongated supramolecular structures has been proposed as a model to explain the generation of fibrillar apatite crystals by protein-guided growth.

Objective: This study is designed to find suitable physical chemical conditions that facilitate hierarchical amelogenin self-assembly from nanospheres into anisometric structures.

Methods: Recombinant amelogenin protein was dissolved at concentration of 1mg/ml in solution at pH 5,6,7, or 8.5 with or without Ca²⁺, PO₄^3-, and KCl. Solutions were left for different time points for self-assembly at 37°C (1,3,5,7 days). Subsequently, proteins were immobilized by placing a 30 µl drop of a 1mg/ml amelogenin solution onto different substrates. Two sets of sample preparations were examined: 1) rinsing off protein solution from glass slide after 1 h, 2) rinsing off glass slide after complete drying of protein. Tapping-mode AFM was used to examine immobilized proteins.

Results: Amelogenin self-assembly into nanospheres of 20 to 30 nm diameter was observed at all pHs used. Elongated structures, including the formation of nanofibers and nanostrings of several hundreds of nanometer length were observed at time points of five and seven days. The addition of Ca²⁺and PO₄^3- ions and low pH increased the probability of fibrillar structure formation through self-assembly.

Conclusion: Hierarchical self-assembly of the full-length human amelogenin, as indicated by the formation of nanostrings and nanofibers is a time-dependent process that requires several days and is enhanced at specific physical chemical conditions.

Support: NIH/NIDCR Grants T32-DE07326 and R01DE17529.