PNNL

Abstract

Amelogenin, the dominant organic component (> 90%) in the early stages of amelogenesis, is an ~180-residue protein that orchestrates the mineralization of apatite crystals into enamel via a poorly understood molecular mechanism. The self-association properties of amelogenin as a function of pH and protein concentration have been suggested to play a central role in guiding the growth of enamel apatite crystals, however, the large molecular weight of the self-assembled quaternary structures have largely prevented structural studies of the protein in solution under physiological conditions. Here, using perdeuterated murine amelogenin (0.25 mM, 5 mg/mL) and TROSY-based NMR experiments to improve spectral resolution, we assigned the 1H-15N spectra of murine amelogenin over a pH range (5.5 to 8.0) where amelogenin is reported to exist as oligomers (pH 7.2). The disappearance or intensity reduction of amide resonances in the 1H-15N HSQC spectra were interpreted to reflect changes in dynamics (intermediate ms-?s motion) and/or heterogenous interfaces of amide nuclei at protein-protein interfaces. The intermolecular interfaces were concentrated towards the N-terminus of amelogenin (L3-G8, V19-G38, L46-Q49, and Q57-L70) at pH 6.6 (oligomers) and at pH 7.2 (nanospheres) included the entire N-terminus up to V73 and regions distributed through the central hydrophobic region (Q82-Q101, S125-Q139 and F151-Q154). At all pHs the C-terminal appeared disordered, highly mobile, and not involved in self-assembly, suggesting nanosphere structures with a solvent-exposed C-termini. Such nanospheres differ from models based on cryoelectron microscopy structures observed on solid mesh surfaces that are composed of dimers stabilized by anti-parallel interactions between C-termini. A concentration titration with 15N-labelled murine ameloginin from 0.05 to 0.5 mM at pH 5.5 mirrored an earlier NaCl titration study at pH 2.8 and showed self-association that was initiated towards the N-terminus and extended to include a region near the C-terminal at high concentration (G8 – S55 and L156 – A170 at 0.5 mM). A pH titration study on an amelogenin construct with eight out of the 13 histidine residues in the hydrophobic region removed suggested the mechanism of amelogenin self-assembly is more complicated than simply changes in the protonation state of histidine residues as a function of pH. These findings present unique, residue-specific insights into the intermolecular protein-protein interfaces driving amelogenin quaternary structure formation and suggest nanospheres in solution predominantly contain disordered, solvent-exposed C-termini.