Following pH-Induced Self-assembly of Murine Amelogenin Using Solution-state NMR Spectroscopy

Objectives: Amelogenesis is a highly coordinated, dynamic, biomineralization process used by vertebrates to make dental enamel. The ~180-residue protein amelogenin is the primary protein responsible for this process. While amelogenin is intrinsically disordered in solution over a wide range of conditions, its oligomerization state varies widely. As the pH is increased, amelogenin transitions from monomers (pH <~4.5) to oligomers (~4.5 to ~6.6) to precipitate (near 7) to nanospheres (>~7.2). Solution-state NMR spectroscopy was used to probe the molecular details of these complexes.
Methods: Murine amelogenin was isotopically labelled with hydrogen-2, carbon-13, and nitrogen-15 using established recombinant protocols. High-resolution ¹H-¹⁵N HSQC spectra were collected on ~0.3 mM samples prepared in low salt buffers adjusted to pH 5.4, 6.6, 7.2, and 8.0. Amides were unambiguously assigned using routine TROSY 3D backbone NMR experiments. Two modified constructs were also prepared (P2C and D180C) to incorporate a paramagnetic probe at each terminus to probe the degree of order in the ensembles.
Results: The major feature of the ¹H-¹⁵N HSQC spectra was the increased broadening, and disappearance, of amide resonances with increasing pH. At pH 6.6 (octomers), amides disappearance was generally localized at three regions: V19-G38, Q57-L70, and H91-Q101. After the transition into nanospheres at pH >7.2, amides from the entire N-terminus up to V73 disappeared along with most residues between Q82-Q101 and H126-Q139. The missing amide resonances likely identify intermolecular interfaces driving the self-assembly of amelogenin.
Conclusions: While the collection and interpretation of the data on the paramagnetic probes are still in progress, a constant feature of the ¹H-¹⁵N HSQC spectra over the pH region surveyed is the presence of the amide resonances for the C-terminal resonances (S157-D180), consistent with models where this C-terminal charged region is exposed on the outside of these complexes to guide calcium phosphate crystallization into hydroxyapatite.