The Role of Basic Amino Acids in the Molecular Recognition of Hydroxyapatite by Statherin using Solid State NMR

Abstract

Organisms use proteins such as statherin to control the growth of hydroxyapatite (HAP), which is the principal component of teeth and bone. Though much emphasis has been placed on the acidic character of these proteins, the role of their basic amino acids is not well understood. In this work, solid state nuclear magnetic resonance was used to probe the interaction of the basic arginine side chains with the HAP surface. Statherin samples were individually labeled at each arginine site, and the distance to the surface was measured using the Rotational Echo DOuble Resonance (REDOR) technique. The results indicate a strong coupling between the R9 and R10 residues and the phosphorus atoms on the surface, with internuclear distances of 4.62 ± 0.29 Å and 4.53 ± 0.16 Å, respectively. Conversely, results also indicate weak coupling between R13 and the surface, suggesting this residue is more removed from the surface than R9 and R10. Combining these results with previous data, a new model for the molecular recognition of HAP by statherin is constructed.

Figure 1

(a) ¹³C CPMAS pulse sequence. (b) XY8 phase cycling ¹³C{³¹P} REDOR pulse sequence with alternating π pulses on both channels. (c) CPP triad model used to simulate the data, with arrows indicating the couplings that were varied in the simulations.

Figure 2

CPMAS spectra of stR9HAP, stR10, and stR13hap acquired at ambient temperature and a spinning rate of 6 kHz along with spectral assignments. Dashed lines correspond to the positions of the labeled FMOC arginine used as the starting material for labeling of all statherin samples. For each spectrum, 50,000 transients were acquired at a repetition rate of 2 s (total acquisition time: 28 hours per sample).

Figure 3

REDOR decay plots for stR9hap, stR10hap, and stR13hap, along with spin triad (CPP) simulations. For the simulations, the ³¹P-³¹P coupling was fixed at 600 Hz, while the two ¹³C-³¹P couplings were varied simultaneously. Between 60,000 and 120,000 transients were acquired at for both the S and S₀ spectra of each data point. The repetition rate was 2 s (total acquisition time: 16 days per sample).

Figure 4

Model of statherin on the HAP surface, combing the new distance constraints outlined in this work with those obtained previously.