The Structure and Orientation of the C-terminus of LRAP
Wendy J Shaw 1*, Kim F Ferris 1, Barbara J Tarasevich 1 and Jenna L Larson 1
1 Pacific Northwest National Labs
* To whom correspondence should be addressed. E-mail: wendy.shaw{at}pnl.gov.
Submitted on August 13, 2007
Revised on October 12, 2007
Accepted on 11 December 2007
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Abstract |
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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 is 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 13C{15N} 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 HAP was investigated for 2 alanine residues (Ala46 and Ala49) using 13C{31P} REDOR and one lysine residue (Lys52) using 15N{31P} REDOR. The residues examined were found to be 7.0, 5.7 and 5.8 Å from the surface of hydroxyapatite, for Ala46, Ala49 and Lys52 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, SSNMR dynamics measurements also revealed significant mobility in the C-terminal region of the protein, both in the sidechains and in the backbone, suggesting that this region alone is not responsible for binding.
Key Words:
amelogenin, biomineralization, enamel, protein structure, solid state NMR
