PNNL

Abstract

Technological developments over the last 10-20 years have made it possible to probe the structure of biomineralization proteins and their interaction with mineral surfaces from the macroscopic to the molecular and atomic level. This knowledge has provided unprecedented insights into the molecular mechanisms responsible for the unique properties of the resulting biominerals. As a model for understanding how biomineralization proteins orchestrate biomineral formation, we have been investigating amelogenin, an ~180-residue intrinsically disordered protein associated with enamel formation. Amelogenin is the predominant matrix protein (90%) secreted in the early stages of the biomineralization process and via the controlled formation of hydroxyapatite (HAP) enamel crystals, amelogeninin contributes to the production of the hardest tissue in the human body, and one of the hardest biominerals in nature. In addition to its intrinsically disordered character, unraveling the role of amelogenin is further compounded by its self-assembly properties that intimately depends on the environment: pH, solutes, solute concentrations, temperature, protein concentration, solution, and surface identity. The heterogeneity and dynamic equilibria of such solutions, coupled with the large molecular weight of the largest self-assembled moieties, nanospheres of 20 – 200 monomers, limited early structural characterization to optical spectroscopies in solution (CD, fluorescence, IR, Raman). These methods provide few molecular details, essentially showing the protein is largely disordered over a wide range of conditions. Thanks to the recent advancements of solution and solid-state NMR spectroscopy, atomic force microscopy (AFM), and recombinant isotope labelling strategies, we are now able to conduct more detailed structural studies with confidence. Our lab has effectively applied NMR spectroscopy to investigate the structural properties of amelogenin in solution and bound to the surface of HAP, its functional form. The solution state NMR studies provided residue specific evidence for amelogenin structural changes due to single point mutations, ionic strength, and pH of the medium. Solid state NMR (ssNMR) studies enabled the first observations of the residue specific structure of amelogenin bound to HAP. This included evidence for interactions between HAP and the N- and C- termini of amelogenin and structural changes between the bound and solubilized protein. AFM provided insight into the thermodynamic forces dictating the quaternary structure of HAP-amelogenin complexes in solution and visual evidence for the presence of protein layers on HAP surfaces. These recent findings, coupled with insights from earlier techniques such as CD and IR spectroscopy and computational methodologies, are contributing to important advancements in our structural understanding of amelogenesis. In this review we focus on recent advances in in situ AFM and solution and solid state NMR spectroscopy that reveal new insights into the secondary, tertiary and quaternary structure of amelogenin by itself and in contact with HAP. These studies have increased our understanding of the interface between amelogenin and HAP and how amelogenin drives the enamel formation mechanism.