Determination of the structure and dynamics of salivary statherin and N-terminal fragments bound to hydroxyapatite using solid state NMR
This dissertation contains solid-state NMR studies of the structure and dynamics of the biomineralization protein, salivary statherin, adsorbed onto its relevant biomineral surface, hydroxyapatite. The structure of statherin and the N-terminal 15 residue peptide were studied using both homonculear and heteronuclear dipolar recoupling techniques. 13C- 13C DRAWS experiments were employed to determine the backbone torsion angle ϕ for samples isotopically labeled at the backbone carbonyl positions at the following residues: pS2pS3, F 7L8 and I11G12, in both the peptide and the full protein. These measurements indicate that the peptide is disordered at the pS2pS3, region with the remaining residues adopting a helical conformation. In contrast, the full protein, studied under hydrated conditions, was found to adopt a well-defined helix throughout most of the pentadecyl domain. The heteronuclear recoupling technique, REDOR, was used to confirm the helical content by measuring the distance between the backbone 13C=O and H15N participating in the hydrogen bond of the alpha-helix. The peptide was labeled at L8G12 and was found to be helical both on and off the surface. The full protein was labeled throughout the pentadecyl region at pS3F7 and L8G12, and agreed with the DRAWS results, supporting a largely helical N-terminus.The dynamics of the peptide and protein under hydrated conditions were studied using T1rho relaxation measurements and line shape analysis. In both cases the pS2pS3, region was found to be very immobile, thus interacting very strongly with the hydroxyapatite surface. The F7L8 region was slightly more mobile in the peptide and even more so in the protein. The I11G12 region experienced a great deal of high frequency and large amplitude motion in the peptide, but had more constrained mobility in the protein. These findings indicate that the highly charged region of the N-terminus is strongly interacting with the surface of hydroxyapatite, indicating the importance of the N-terminal pentapeptide in the binding of statherin, while the remainder of the domain is only weakly interacting with the surface.
- Chemistry