Energetics of Amelogenin Binding to Hydroxyapatite – Insights into Amelogenesis Imperfecta

Objectives: The exceptional functional properties of enamel, one of nature’s hardest materials, arise from its intricate hierarchical structure and cannot be reproduced in vitro inorganically. In vivo the formation of enamel depends on the ~180-residue protein amelogenin, the predominant protein in the enamel matrix. A testament to amelogenin’s importance is that a single amino acid substitution to the primary amino acid sequence of amelogenin can lead to drastic changes in enamel phenotype, resulting in amelogenesis imperfecta (AI), enamel that is defective and easily damaged. Our aim was to develop a thermodynamic understanding to explain how a single amino acid variation can lead to malformed enamel.
Methods:
High resolution, in situ atomic force microscopy (AFM) was employed to study the energetics of murine amelogenin (wild-type, two naturally occurring single amino acid variants associated with AI (T21I and P41T), and a model variant (P71T)) binding onto single crystal hydroxyapatite (100) in real time.
Results: In situ AFM showed that altering one amino acid resulted in an increase in the quantity of protein adsorbed onto hydroxyapatite and the formation of multiple protein layers. Quantitative analysis of the equilibrium adsorbate amounts revealed that the protein variants had higher protein-protein binding energies. Hydroxyapatite mineralization and MMP20 enzyme degradation studies showed that the amino acid variants inhibited the growth and phase transformation of hydroxyapatite and slowed the degradation of amelogenin by MMP20.
Conclusions: The amelogenin variants cause malformed enamel because they bind excessively to hydroxyapatite and disrupt the normal hydroxyapatite growth and enzymatic degradation processes. The in situ methods we applied to determine the molecular level energetics of amelogenin binding to hydroxyapatite are powerful tools to be further exploited towards understanding the mechanisms of biomineralization. The ultimate result of such understanding is the predictive synthesis of functional materials tailored by macromolecular design.