Proteins play an important role in inorganic crystal engineering during the development and growth of hard tissues such as bone and teeth. Although many of these proteins have been studied in the liquid state, there is little direct information describing molecular recognition at the protein-crystal interface. Here we have used 13 C solid-state NMR (SSNMR) techniques to investigate the conformation of an N-terminal peptide of salivary statherin both free and adsorbed on hydroxyapatite (HAP) crystals. The torsion angle φ was determined at three positions along the backbone of the phosphorylated N-terminal 15 amino acid peptide fragment (DpSpSEEKFLRRIGRFG) by measuring distances between the backbone carbonyls carbons in the indicated adjacent amino acids using dipolar recoupling with a windowless sequence (DRAWS). Global secondary structure was determined by measuring the dipolar coupling between the 13 C backbone carbonyl and the backbone 15 N in the i f i + 4 residues (DpSpSEEKFLRRIGRFG) using rotational echo double resonance (REDOR). Peptides singly labeled at amino acids pS 3 , L 8 , and G 12 were used for relaxation and line width measurements. The peptides adsorbed to the HAP surface have an average φ of -85°at the N-terminus (pSpS), -60°in the middle (FL) and -73°near the C-terminus (IG). The average φ angle measured at the pSpS position and the observed high conformational dispersion suggest a random coil conformation at this position. However, the FL position displays an average φ that indicates significant R-helical content, and the long time points in the DRAWS experiment fit best to a relatively narrow distribution of φ that falls within the protein data bank R-helical conformational space. REDOR measurements confirm the presence of helical content, where the distance across the LG hydrogen bond of the adsorbed peptide has been found to be 5.0 Å. The φ angle measured at the IG position falls at the upper end of the protein data bank R-helical distribution, with a best fit to a relatively broad φ distribution that is consistent with a distribution of R-helix and more extended backbone conformation. These results thus support a structural model where the N-terminus is disordered, potentially to maximize interactions between the HAP surface and the negatively charged side chains found in this region, the middle portion is largely R-helical, and the C-terminus has a more extended conformation (or a mixture of helix and extended conformations).

Proteins play an important role in the biological mechanisms controlling hard tissue development, but the details of molecular recognition at inorganic crystal interfaces remain poorly characterized. We have applied a recently developed homonuclear dipolar recoupling solidstate NMR technique, dipolar recoupling with a windowless sequence (DRAWS), to directly probe the conformation of an acidic peptide adsorbed to hydroxyapatite (HAP) crystals. The phosphorylated hexapeptide, DpSpSEEK (N6, where pS denotes phosphorylated serine), was derived from the N terminus of the salivary protein statherin. Constantcomposition kinetic characterization demonstrated that, like the native statherin, this peptide inhibits the growth of HAP seed crystals when preadsorbed to the crystal surface. The DRAWS technique was used to measure the internuclear distance between two 13 C labels at the carbonyl positions of the adjacent phosphoserine residues. Dipolar dephasing measured at short mixing times yielded a mean separation distance of 3.2 ؎ 0.1 Å. Data obtained by using longer mixing times suggest a broad distribution of conformations about this average distance. Using a more complex model with discrete ␣-helical and extended conformations did not yield a better fit to the data and was not consistent with chemical shift analysis. These results suggest that the peptide is predominantly in an extended conformation rather than an ␣-helical state on the HAP surface. Solid-state NMR approaches can thus be used to determine directly the conformation of biologically relevant peptides on HAP surfaces. A better understanding of peptide and protein conformation on biomineral surfaces may provide design principles useful for the modification of orthopedic and dental implants with coatings and biological growth factors that are designed to enhance biocompatibility with surrounding tissue.