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

ABSTRACT: 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.

SUBMITTER: Buchko GW

PROVIDER: S-EPMC10286517 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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

Buchko Garry W GW Mergelsberg Sebastian T ST Tarasevich Barbara J BJ Shaw Wendy J WJ

Biochemistry 20221201 24

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 conventiona ...[more]

PMID: 36456190

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Project description:Because self-assembly of matrix proteins is a key step in hard tissue mineralization, developing an understanding of the assembly pathways and underlying mechanisms is likely to be important for successful hard tissue engineering. While many studies of matrix protein assembly have been performed on bulk solutions, in vivo these proteins are likely to be in contact with charged biological surfaces composed of lipids, proteins, or minerals. Here we report the results of an in situ atomic force microscopy (AFM) study of self-assembly by amelogenin--the principal protein of the extracellular matrix in developing enamel--in contact with two different charged substrates: hydrophilic negatively charged bare mica and positively charged 3-aminopropyl triethoxysilane (APS) silanized mica. First we demonstrate an AFM-based protocol for determining the size of both amelogenin monomers and oligomers. Using this protocol, we find that, although amelogenin exists primarily as ~26 nm in diameter nanospheres in bulk solution at a pH of 8.0 studied by dynamic light scattering, it behaves dramatically differently upon interacting with charged substrates at the same pH and exhibits complex substrate-dependent assembly pathways and dynamics. On positively charged APS-treated mica surfaces, amelogenin forms a relatively uniform population of decameric oligomers, which then transform into two main populations: higher-order assemblies of oligomers and amelogenin monomers, while on negatively charged bare mica surfaces, it forms a film of monomers that exhibits tip-induced desorption and patterning. The present study represents a successful attempt to identify the size of amelogenin oligomers and to directly monitor assembly and disassembly dynamics on surfaces. The findings have implications for amelogenin-controlled calcium phosphate mineralization in vitro and may offer new insights into in vivo self-assembly of matrix proteins as well as their control over hard tissue formation.

Dataset Information

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

Publications

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

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