The structure and orientation of the C-terminus of LRAP.
Amelogenin is the predominant protein found during enamel development and is thought to be the biomineralization protein controlling the unique elongated hydroxyapatite crystals that constitute enamel. The secondary structure of biomineralization proteins is thought to be important in the interaction with hydroxyapatite. Unfortunately, very little data are available on the structure or the orientation of amelogenin, either in solution or bound to hydroxyapatite. The C-terminus contains the majority of the charged residues and is predicted to interact with hydroxyapatite; thus, we used solid-state NMR dipolar recoupling techniques to investigate the structure and orientation of the C-terminus of LRAP, a naturally occurring splice variant of full-length amelogenin. Using (13)C{(15)N} Rotational Echo DOuble Resonance (REDOR), the structure of the C-terminus was found to be largely random coil, both on the surface of hydroxyapatite as well as lyophilized from solution. The orientation of the C-terminal region with respect to hydroxyapatite was investigated for two alanine residues (Ala(46) and Ala(49)) using (13)C{(31)P} REDOR and one lysine residue (Lys(52)) using (15)N{(31)P} REDOR. The residues examined were found to be 7.0, 5.7, and 5.8 A from the surface of hydroxyapatite for Ala(46), Ala(49), and Lys(52), respectively. This provides direct evidence that the charged C-terminus is interacting closely with hydroxyapatite, positioning the acidic amino acids to aid in controlling crystal growth. However, solid-state NMR dynamics measurements also revealed significant mobility in the C-terminal region of the protein, in both the side chains and the backbone, suggesting that this region alone is not responsible for binding.
Shaw
Wendy J
WJ
Pacific Northwest National Laboratory, Richland, Washington 99352, USA. wendy.shaw@pnl.gov
Ferris
Kim
K
Tarasevich
Barbara
B
Larson
Jenna L
JL
D003198:Computer Simulation; D003746:Dental Enamel Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D008956:Models, Chemical; D008958:Models, Molecular; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
D003746:Dental Enamel Proteins; C067432:leucine-rich amelogenin peptide