Abstract
Amelogenin, the predominant extracellular matrix protein secreted by ameloblasts, has been shown to be essential for proper tooth enamel formation. In this study, amelogenin adsorption to hydroxyapatite (HAP) surfaces, a prototype for enamel mineral, has been studied using a quartz crystal microbalance (QCM) to interrogate effects of protein phosphorylation and solution pH. Dynamic flow-based experiments were conducted at pH 7.4 and 8.0 using native phosphorylated porcine amelogenin (P173) and recombinant non-phosphorylated porcine amelogenin (rP172). Loading capacities (μmol/m2) on HAP surfaces were calculated under all conditions and adsorption affinities (Kad) were calculated when Langmuir isotherm conditions appeared to be met. At pH 8.0, binding characteristics were remarkably similar for the two proteins. However, at pH 7.4 a higher affinity and lower surface loading for the phosphorylated P173 was found compared to any other set of conditions. This suggests that phosphorylated P173 adopts a more extended conformation than non-phosphorylated full-length amelogenin, occupying a larger footprint on the HAP surface. This surface-induced structural difference may help explain why P173 is a more effective inhibitor of spontaneous HAP formation in vitro than rP172. Differences in the viscoelastic properties of P173 and rP172 in the adsorbed state were also observed, consistent with noted differences in HAP binding. These collective findings provide new insight into the important role of amelogenin phosphorylation in the mechanism by which amelogenin regulates enamel crystal formation.