Sarah Burton1 Rajith Jayasinha Arachchige1 Garry Buchko1 Bojana Ginovska1 Larissa Harding1 Jinhui Tao1 Barbara Tarasevich1 Wendy Shaw1

1, Pacific Northwest National Laboratory, Richland, Washington, United States

Enamel is consider as one of nature’s hardest materials. It is formed extracellularly by a complex process involving multiple proteins. The most common protein present during enamel formation is amelogenin (>90%). Amelogenin’s dominance in the extracellular matrix along with a multitude of both in vivo and in vitro studies demonstrate that it can strongly influence the shape and properties of the resulting crystals. This influence is so controlled, that even single amino acid modifications in the primary structure can result in severely dysfunctional enamel. While amelogenin’s role in enamel formation is widely agreed upon, the mechanism of growth and control at a molecular level is less clear. Studies by Beniash and coworkers have proposed a growth mechanism based on cryo-EM studies which identify amelogenin dimers as a design group upon which the larger hierarchical structures are built.
In this work, we extend the use of solid state NMR to study large biomineralization proteins a step further, to probe intermolecular interactions which dominate the proposed secondary structure of this protein. Based on the overlapping tail-to-tail interactions of amelogenin dimers in the presence of calcium phosphate deduced by the cryo-EM study, we propose that the dimer conformations are stabilized by salt bridges. We selected a specific stable isotope labeling (13C and 15N) scheme to probe this bridging interaction that fit two criteria: 1) residues that do not “scramble” excessively during biological synthesis, i.e. the biosynthetic pathway does not result in additional residues being labeled; and 2) residues that should be close enough in space to detect if salt bridges are present. Using detail 2D-solid state NMR study, we have identified a unique atomic level intermolecular interaction stabilizing amelogenin dimers. The demonstration of atomic level intermolecular interactions for a biomineralization protein bound to its biologically relevant surface is an important advancement in this field and allows us to further define mechanistic details for the formation of hard tissues such as enamel.