Determination of Statherin N-Terminal Peptide Conformation on Hydroxyapatite Crystals

Abstract

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 13C 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 13C backbone carbonyl and the backbone 15N in the i → i + 4 residues (DpSpSEEKFLRRIGRFG) using rotational echo double resonance (REDOR). Peptides singly labeled at amino acids pS3, L8, and G12 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 α-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 α-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 α-helical distribution, with a best fit to a relatively broad φ distribution that is consistent with a distribution of α-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 α-helical, and the C-terminus has a more extended conformation (or a mixture of helix and extended conformations).