Residue-Specific Insights into the Intermolecular Protein–Protein Interfaces Driving Amelogenin Self-Assembly in Solution

• Published in: Biochemistry (Easton) Vol. 61; no. 24; pp. 2909 - 2921
• Main Authors: Buchko, Garry W., Mergelsberg, Sebastian T., Tarasevich, Barbara J., Shaw, Wendy J.
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 20.12.2022
• Subjects: Amelogenin - chemistry; Amelogenin - metabolism; Amides; Animals; Dental Enamel Proteins; Magnetic Resonance Spectroscopy; Mice; Solvents
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/acs.biochem.2c00522

Amelogenin, the dominant organic component (>90%) in the early stages of amelogenesis, orchestrates the mineralization of apatite crystals into enamel. The self-association properties of amelogenin as a function of pH and protein concentration have been suggested to play a central role in this process; however, the large molecular weight of the self-assembled quaternary structures has largely prevented structural studies of the protein in solution under physiological conditions using conventional approaches. 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 ≤∼6.8) and nanospheres (pH ≥∼7.2). The disappearance or intensity reduction of amide resonances in the 1H-15N HSQC spectra was interpreted to reflect changes in dynamics (intermediate millisecond-to-microsecond motion) and/or heterogenous interfaces of amide nuclei at protein–protein interfaces. The intermolecular interfaces were concentrated toward 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) including the entire N-terminus up to Q76 and regions distributed through the central hydrophobic region (Q82-Q101, S125-Q139, and F151-Q154). At all pH levels, the C-terminus appeared disordered, highly mobile, and not involved in self-assembly, suggesting nanosphere structures with solvent-exposed C-termini. These findings present unique, residue-specific insights into the intermolecular protein–protein interfaces driving amelogenin quaternary structure formation and suggest that nanospheres in solution predominantly contain disordered, solvent-exposed C-termini.