Solid-State NMR Identification of Intermolecular Interactions in Amelogenin Bound to Hydroxyapatite

Abstract

Biomineralization processes govern the formation of hierarchical hard tissues such as bone and teeth in living organisms, and mimicking these processes could lead to the design of new materials with specialized properties. However, such advances require structural characterization of the proteins guiding biomineral formation to understand and mimic their impact. In their ''active'' form, biomineralization proteins are bound to a solid surface, severely limiting our ability to use many conventional structure characterization techniques. Here, solid-state NMR spectroscopy was applied to study the intermolecular interactions of amelogenin, the most abundant protein present during the early stages of enamel formation, in self-assembled oligomers bound to hydroxyapatite. Intermolecular dipolar couplings were identified that support amelogenin dimer formation stabilized by residues toward the C-termini. These dipolar interactions were corroborated by molecular dynamics simulations. A b-sheet structure was identified in multiple regions of the protein, which is otherwise intrinsically disordered in the absence of hydroxyapatite. To our knowledge, this is the first intermolecular protein-protein interaction reported for a biomineralization protein, representing an advancement in understanding enamel development and a new general strategy toward investigating biomineralization proteins.

Backbone chemical shift assignments for the SARS-CoV-2 non-structural protein Nsp9: intermediate (ms – μs) dynamics in the C-terminal helix at the dimer interface

“…The cleaved protein, which contained three N-terminal scar residues afterwards (GHM-), was purified by re-application on the size exclusion column. To assist amide assignments, residue-specific, 15 N-labeled Leu, Val, Phe, Lys, and Met samples were prepared using a modified “Redfield-medium” (Arachchige et al 2018 ) and the protein purified as described above.…”

NMR Methods for Structural Characterization of Protein-Protein Complexes

“…Using perdeuteration or ultrafast MAS, 1 H assignments can also be obtained. Therefore, MAS NMR facilitates the atomic-level analysis of protein-protein interactions in biological solids (Marulanda et al, 2004;Miao and Cross, 2013;Arachchige et al, 2018;van der Wel, 2018). Systems where only one of the two interacting proteins is natively found in the solid-state, as is the case with membrane proteins that interact with soluble proteins as part of a signal transduction pathway, are also amenable to solid-state NMR analysis.…”

Structural Biology of Calcium Phosphate Nanoclusters Sequestered by Phosphoproteins

“…Other motifs include the CaP-SLiMs and short cationic or anionic sequences that promote attractive, electrostatic, protein-protein interactions [28]. Through a range of different types of interactions mediated by their SLIMs, the non-SIBLING SCPPs have a tendency to associate to form homotypic or heterotypic oligomers or larger assemblies such as gels or amyloid fibrils and ribbons [28][29][30][31][32][33][34].…”