The energetic basis for hydroxyapatite mineralization by amelogenin variants provides insights into the origin of amelogenesis imperfecta

Abstract

Small variations in the primary amino acid sequence of extracellular matrix proteins can have profound effects on the biomineralization of hard tissues. For example, a change in one amino acid within the amelogenin protein can lead to drastic changes in enamel phenotype, resulting inamelogenesis imperfecta, enamel that is defective and easily damaged. Despite the importance of these undesirable phenotypes, there is very little understanding of how single amino acid variation in amelogenins can lead to malformed enamel. Here, we aim to develop a thermodynamic understanding of how protein variants can affect steps of the biomineralization process. High-resolution, in situ atomic force microscopy (AFM) showed that altering one amino acid within the murine amelogenin sequence (natural variants T21 and P41T, and experimental variant P71T) resulted in an increase in the quantity of protein adsorbed onto hydroxyapatite (HAP) and the formation of multiple protein layers. Quantitative analysis of the equilibrium adsorbate amounts revealed that the protein variants had higher oligomer–oligomer binding energies. MMP20 enzyme degradation and HAP mineralization studies showed that the amino acid variants slowed the degradation of amelogenin by MMP20 and inhibited the growth and phase transformation of HAP. We propose that the protein variants cause malformed enamel because they bind excessively to HAP and disrupt the normal HAP growth and enzymatic degradation processes. The in situ methods applied to determine the energetics of molecular level processes are powerful tools toward understanding the mechanisms of biomineralization.

Molecular Dynamics Characterization of Sr-Doped Biomimetic Hydroxyapatite Nanoparticles

“…Amino acids, as basic units of proteins, have some important influences on the adsorption and recognition of implanted biomaterial surfaces. , The substitution of apatite implants may affect the interaction between the material surface and adsorbents, thus affecting the related bone metabolism. Glu is a representative negatively charged amino acid whose side chain is carboxylate.…”

On Simulating the Formation of Structured, Crystalline Systems via Non-classical Pathways

“…These direct observations enable for the investigation of events immediately before and after nucleation as well as during crystal growth in order to observe intermediate and "exotic" states altering particle dynamics that are difficult to observe on smaller scales and only now becoming noticed as important for driving growth along specific pathways (Gebauer et al, 2008;Dey et al, 2010;Picker et al, 2017). Of specific interest are questions related to amorphous precursors, neighboring crystal domains, and their effects on the resulting material structure as observed by previous work in biological materials and systems (Aizenberg, 2004;Politi et al, 2004;Munch et al, 2008;Meyers et al, 2013;Fischer et al, 2017;Tao et al, 2019). Similar model systems of attractive microbeads have revealed that island formation, step-edge barriers (Ganapathy et al, 2010), and even multi-layer growth on heavily strained substrates (Lechner et al, 2011;Savage et al, 2013) arise even when the length scale of the interparticle interaction is far shorter than a particle radius.…”

“…For example, crystal growth assisted by structured pre-crystalline states (Gebauer et al, 2008;Dey et al, 2010;Demichelis et al, 2011), nucleation from metastable phases (Chung et al, 2009;Washington et al, 2012;Baumgartner et al, 2013;Maes et al, 2015), oriented attachment of crystallites (Banfield et al, 2000;Li et al, 2012;Nielsen et al, 2014), and growth emerging out of amorphous phases (Weiss et al, 2002;Politi et al, 2004;Killian et al, 2009;Savage and Dinsmore, 2009;Mahamid et al, 2010;Lechner et al, 2011;Salvalaglio et al, 2014;Ma et al, 2017;Pendola et al, 2018) have all been shown to alter growth in non-trivial ways, often resulting in complex structure formation that appear to defy the evolution of the system toward its ultimate lowest energy configuration. Moreover, these types of alternative growth mechanisms have been suggested in systems with widely different environmental conditions, from biological context such as protein crystal nucleation (Vekilov and Vorontsova, 2014), calcite growth (Weiss et al, 2002;Politi et al, 2004;Killian et al, 2009;, tissue mineralization (Wang et al, 2012;Weaver et al, 2012;Tao et al, 2019), magnetite nucleation and growth (Kuhrts et al, 2019;Mirabello et al, 2019;Rawlings et al, 2019), as well as inorganic contexts like cadmium selenide quantum dot growth (Washington et al, 2012), iron oxide growth (Banfield et al, 2000;Baumgartner et al, 2013;Dideriksen et al, 2015), and colloidal microparticle crystallization (Savage et al, 2...…”

Diffusion-Controlled Crystallization of Calcium Phosphate in a Hydrogel toward a Homogeneous Octacalcium Phosphate/Agarose Composite

“…Sol 0.5 /Gel 0.3 and Sol 0.083 /Gel 0.05 composite systems were composed of a 0.5 M CaCl 2 solution (Sol 0.5 ), a 0.3 M Na 2 HPO 4 hydrogel (Gel 0.3 ), a 0.083 M CaCl 2 solution (Sol 0.083 ), and a 0.05 M Na 2 HPO 4 hydrogel (Gel 0.05 ), respectively. The 1.67:1 ratio of Ca 2+ and HPO 4 2– was chosen based on the stoichiometry of hydroxyapatite (Ca 5 (PO 4 ) 3 OH), which has been widely used for the synthesis of various types of calcium phosphate in previous reports. − The Gel 0.5 /Gel 0.3 composite system was composed of a 0.5 M CaCl 2 hydrogel (Gel 0.5 ) and a 0.3 M Na 2 HPO 4 hydrogel. In the Sol 0.5 /Gel/Gel 0.3 composite system, there was an additional hydrogel layer (Gel) that contained no electrolyte between the 0.5 M CaCl 2 solution and the 0.3 M Na 2 HPO 4 hydrogel.…”