Solid-State NMR Captures Time-Resolved Conformational Remodeling of Amelogenin Assemblies in Guiding Hydroxyapatite...

Amelogenin is a major component in developing enamel protein matrix and plays a key role in regulating enamel mineral morphology. However, intrinsic disorder encoded in amelogenin's low-complexity regions leads to structural heterogeneity, thereby evading high-resolution characterization under physiological conditions. We hypothesize that amelogenin undergoes domain-specific rather than uniform structural and dynamic remodeling in regulating enamel apatite formation. Solid-state NMR (SSNMR), coupled with complementary techniques including scanning electron microscopy (SEM), AFM-based nano-scale Infrared Spectroscopy (AFM-NanoIR), X-ray diffraction (XRD), transmission electron microscopy (TEM), was employed to correlate amelogenin structural transitions with the mineral morphological changes across atomic to microscale resolutions at near physiological conditions in this study. Both cross-polarization and J-coupling based magic-angle-spinning (MAS) SSNMR experiments delineated an assembly landscape comprising rigid and dynamic components during calcium phosphate mineralization. Time-resolved tracking revealed distinct, stage-specific domain behaviors: the N-terminal domain partially adopts a β-strand secondary conformation with amyloid properties and gradually gains ordering towards the late-stage biomineralization, coinciding with HAP crystal elongation; the C-terminal domain maintains structurally rigidity but with structural reorganization during the mineralization; the central domain becomes increasingly dynamic as mineralization progresses, indicated by J-coupling-based 3D NMR experiments (HNCA, HNcoCA, HNCO, and CBCAcoNH), suggesting interactions with the minerals earlier, but later weakened protein-mineral contacts. These domain-specific, spatiotemporal roles collectively may contribute to the hierarchical mineralization process, providing insight into enamel formation. STATEMENT OF SIGNIFICANCE: Understanding how nature builds complex materials like tooth enamel requires tracking the dynamic role of proteins, which are often transient in nature. By integrating advanced solid-state NMR with high-resolution imaging, we captured, for the first time, how the disordered protein amelogenin remodels itself over time to guide the growth of hydroxyapatite crystals. We discovered that the protein's N-terminal region becomes rigid to form amyloid-like scaffolds, which is associated to the crystal elongation in a timely manner, while the central region becomes mobile. This unprecedented, time-resolved view reveals how a dynamic protein matrix orchestrates hierarchical mineralization, providing a blueprint for bio-inspired synthesis of hard tissues and regenerative dental therapies.