A ¹³C{ ³¹P} REDOR NMR investigation of the role of glutamic acid residues in statherin- Hydroxyapatite recognition

The side chain carboxyl groups of acidic proteins found in the extra-cellular matrix (ECM) of mineralized tissues play a key role in promoting or inhibiting the growth of minerals such as hydroxyapatite (HAP), the principal mineral component of bone and teeth. Among the acidic proteins found in the saliva is statherin, a 43-residue tyrosine-rich peptide that is a potent lubricant in the salivary pellicle and an inhibitor of both HAP crystal nucleation and growth. Three acidic amino acids;D1, E4, and E5;are located in the N-terminal 15 amino acid segment, with a fourth amino acid, E26, located outside the N-terminus. We have utilized ¹³C{ ³¹P} REDORNMRto analyze the role played by acidic amino acids in the binding mechanism of statherin to theHAPsurface by measuring the distance between the δ-carboxyl ¹³C spins of the three glutamic acid side chains of statherin (residues E4, E5, E26) and ³¹P spins of the phosphate groups at the HAP surface. ¹³C{ ³¹P} REDOR studies of glutamic-5- ¹³C acid incorporated at positions E4 and E26 indicate a ¹³C- ³¹P distance of more than 6.5 Å between the side chain carboxyl ¹³C spin of E4 and the closest ³¹P in the HAP surface. In contrast, the carboxyl ¹³C spin at E5 has a much shorter ¹³C- ³¹P internuclear distance of 4.25 ± 0.09 Å , indicating that the carboxyl group of this side chain interacts directly with the surface. ¹³C T ₁ε and slow-spinning MAS studies indicate that the motions of the side chains of E4 and E5 are more restricted than that of E26. Together, these results provide further insight into the molecular interactions of statherin with HAP surfaces.