A PEPTIDE THAT INHIBITS HYDROXYAPATITE GROWTH IS IN AN EXTENDED CONFORMATION ON THE CRYSTAL-SURFACE

Proteins play an important role in the biological mechanisms controlli ng hard tissue development, but the details of molecular recognition a t inorganic crystal interfaces remain poorly characterized. We have ap plied a recently developed homonuclear dipolar recoupling solid-state NMR technique, dipolar recoupling with a windowless sequence (DRAWS), to directly probe the conformation of an acidic peptide adsorbed to hy droxyapatite (HAP) crystals. The phosphorylated hexapeptide, DpSpSEEK (N6, where pS denotes phosphorylated serine), was derived from the N t erminus of the salivary protein statherin, Constant-composition kineti c characterization demonstrated that, like the native statherin, this peptide inhibits the growth of HAP seed crystals when preadsorbed to t he crystal surface. The DRAWS technique was used to measure the intern uclear distance between two C-13 labels at the carbonyl positions of t he adjacent phosphoserine residues. Dipolar dephasing measured at shor t mixing times yielded a mean separation distance of 3.2 +/- 0.1 Angst rom. Data obtained by using longer mixing times suggest a broad distri bution of conformations about this average distance. Using a more comp lex model with discrete cy-helical and extended conformations did not yield a better fit to the data and was not consistent with chemical sh ift analysis, These results suggest that the peptide is predominantly in an extended conformation rather than an Lu-helical state on the HAP surface, Solid-state NMR approaches can thus be used to determine dir ectly the conformation of biologically relevant peptides on HAP surfac es. A better understanding of peptide and protein conformation on biom ineral surfaces may provide design principles useful for the modificat ion of orthopedic and dental implants with coatings and biological gro wth factors that are designed to enhance biocompatibility with surroun ding tissue.