A peptide that inhibits hydroxyapatite growth is in an extended conformation on the crystal surface

Abstract

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.

MEPE has the properties of an osteoblastic phosphatonin and minhibin

“…Statherin, a salivary protein, is a 62-residue molecule with asymmetric charge and structural properties. A key role is maintaining the mineral solution dynamics of enamel by virtue of its ability to inhibit spontaneous precipitation and crystal growth from supersaturated solutions of calcium phosphate minerals [5,43,64,78]. Statherin's role in preserving the calcium phosphate supersaturated state of saliva is crucial for re-calcification and stabilization of tooth enamel and for the inhibition of formation of mineral accretions on tooth surfaces.…”

“…2) originally identified by us [71]. Moreover, this peptide has been implicated in inhibiting calcification of the urinary tract [32,96] and the motif is also present in salivary statherin and is responsible for maintaining the phosphate/calcium supersaturated mineralization solution dynamics of saliva [5,43,64,78]. Statherin may also function in the transport of calcium and phosphate during secretion in the salivary glands [5,64,78].…”

Solid state NMR studies of molecular recognition at protein–mineral interfaces

“…Analysis of the magnetization decay both in the reference experiment (S 0 ) to obtain homonuclear dipolar couplings and in the normalized dephasing experiment (S/S 0 ) to derive heteronuclear dipolar couplings yields complete characterization of the spin triad and enables us to extend accurate structural information deduced from selective labeled sample. Dipolar recoupling with a windowless sequence (DRAWS) is well suited to measuring distances between backbone carbonyl carbons in peptides and proteins [37][38][39][40]. Dipolar couplings of less than 40 Hz between carbonyl 13 C spins have been detected in biopolymers at high magnetic fields [41][42][43][44], attesting to the ability of DRAWS to suppress large CSAs and at the same time detect small dipolar couplings between non-bonded 13 C spins.…”

“…They exhibit an average pair RMSD of 2.5 Å with higher structural dispersion in the intermediate region where constraints have not yet been measured. The small folded core is created by the folding of the C-terminus helical region (33)(34)(35)(36)(37)(38) back onto the intermediate region through a loop region defined by proline residues. The C-terminus helix can be tilted at different angles with respect to the N-terminus helix due to the limited number of constraints between the two regions.…”

“…These models are derived from the full set of 1,000 models. Alignment of the models in the figure was based on root-mean-square deviation calculation of segments (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) and (33)(34)(35)(36)(37)(38) between the 8 models. Table 1 31 P-31 P nearest neighbor distances lying within a sphere of radius 6 Å for the 004 crystal plane in in monoclinic HAP.…”