PNNL

Abstract

Amelogenin is the predominant protein found during enamel development. The formation of amelogenin into nanosphere quaternary structures is thought to control the unique elongated hydroxyapatite crystals that constitute enamel. Consequently, the secondary structure of the protein will be important in the formation of the nanosphere, as well as in the interaction with hydroxyapatite, by revealing or concealing critical amino acids. Unfortunately, very little data is available on the structure or the orientation of the protein, either in solution or bound to hydroxyapatite. Here, we used solid state NMR techniques to investigate the structure and orientation of the C-terminus of LRAP, a naturally occurring splice variant of full length amelogenin. The structure of the C-terminus showed a largely random coil or extended structure, both on the surface of hydroxyapatite as well as lyophilized from solution. The orientation of the C-terminal region with respect to HAP was investigated for 2 alanine residues (Ala46 and Ala49) and one lysine residue (Lys52). The sidechain terminal methyl or amine group of the residues examined were found to be 7, 6 and 6 Å from the surface of hydroxyapatite, for Ala46 Ala49 and Lys52 respectively, providing direct evidence that the charged C-terminus is interacting closely with hydroxyapatite. However, the C-terminal region was also found to have medium to large frequency and amplitude dynamics, both in the sidechains and in the backbone, suggesting that this region alone is not responsible for binding. The data is discussed in terms of a binding mechanism. This work was supported by NIH-NIDCR Grant DE-015347 and was performed at Pacific Northwest National Laboratory, operated by Battelle for the US-DOE.