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

Abstract

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 (13)C{(31)P} REDOR NMR to analyze the role played by acidic amino acids in the binding mechanism of statherin to the HAP surface by measuring the distance between the delta-carboxyl (13)C spins of the three glutamic acid side chains of statherin (residues E4, E5, E26) and (31)P spins of the phosphate groups at the HAP surface. (13)C{(31)P} REDOR studies of glutamic-5-(13)C acid incorporated at positions E4 and E26 indicate a (13)C-(31)P distance of more than 6.5 A between the side chain carboxyl (13)C spin of E4 and the closest (31)P in the HAP surface. In contrast, the carboxyl (13)C spin at E5 has a much shorter (13)C-(31)P internuclear distance of 4.25 +/- 0.09 A, indicating that the carboxyl group of this side chain interacts directly with the surface. (13)C T(1rho) 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.

Figure 1

Structure of (a) glutamic acid, 5-¹³C, (b) Fmoc-glutamic acid (5-¹³C) t-butyl ester. ¹³C labeled site indicated with asterisk.

Figure 2

XY8 phase cycling ¹³C{³¹P} REDOR pulse sequence with alternating π pulses.

Figure 3

³¹P spin topologies used in simulation of ¹³C{³¹P} REDOR data. Arrows in the figure indicate the dipolar couplings that were varied in simulations. Homonuclear coupling of pseudo-³¹P-pair was fixed at 600 Hz based on previous studies.

Figure 4

CPMAS spectra of stE4hap, stE5hap, and polycrystalline glutamic acid (E) acquired at room temperature and a spinning rate of 4 kHz.

Figure 5

REDOR decay plots for the three statherin samples bound to HAP. Circles indicate stE4hap, squares indicate stE5hap, and triangles indicate stE26hap.

Figure 6

Simulated ¹³C{³¹P} REDOR curves for three possible spin systems: (a) 2-spin ¹³C–³¹P coupling, (b) 3-spin coupling of one ¹³C to two ³¹P nuclei without any ³¹P–³¹P coupling, (c) 3-spin ¹³C–³¹P–³¹P coupling with fixed 600 Hz ³¹P–³¹P coupling. Experimental stE5hap data (square symbols) are depicted in (a) – (c) as well. (d) through (f) are the corresponding χ² values calculated and plotted based on simulations carried at each value of the C–P internuclear distance (Å) shown by the triangle symbols.

Figure 7

HAP-bound statherin T_1ρ values for E4, E5, and E26 as functions of sample temperature.

Figure 8

Model of N-terminal region of statherin on HAP surface, combining new and previously established distance constraints.