A REDOR NMR Study of a Phosphorylated Statherin Fragment Bound to Hydroxyapatite Crystals

Abstract

Hydroxyapatite (HAP) is the main mineral component of teeth. It is well-known that several salivary proteins and peptides bind strongly to HAP to regulate crystal growth. Interactions between a peptide derived from the N-terminal fragment of the salivary protein statherin and HAP were measured utilizing rotational-echo double-resonance (REDOR) nuclear magnetic resonance (NMR). The REDOR measurement from the side chain of the salivary peptide to the HAP surface is complicated by two effects: a possible additional dipolar coupling to a phosphorylated side chain and the potential proximity of phosphorus atoms to each other, resulting in a homonuclear dipolar interaction. Both of these effects were addressed, and the smallest model applicable to our system includes the nitrogen-15 (15N) spin in the lysine side chain and two phosphorus-31 (31P) spins, at least one of which must be from the surface phosphates of the HAP.

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

“…Statherin, a small 43 amino acid protein, readily binds calcium ions in solution and adsorbs to HAP surfaces with a significant binding affinity and coverage [53][54][55]. The proximity of statherin side chains to the surface and the mechanism of adsorption to HAP were recently investigated [55][56][57]. The viscoelastic properties of statherin, PRPs and mucins in the salivary pellicles reduce by 20-fold the mastication load [50,58].…”

“…Distance measurements between side chains on the SN-15 derivative and NMR active 31 P atoms on the HAP surface were used to learn about the proximity of particular residues to the surface. NP-REDOR measurements were carried out on a 15 Nε(K6) labeled SN15 sample [57,86]. Analysis of REDOR measurements in the case where an atom on the protein might be coupled to several phosphate groups on the surface was detailed in two publications [57,87].…”

Multilayered core–shell structure of polyol-stabilized calcium fluoride nanoparticles characterized by NMR

“…NMR has been increasingly applied over the last few years to investigate the core-surface, core-shell and interface/interphase properties of nanoscale compounds [15,16]. Examples include inner surfaces of zeolites [17][18][19][20][21][22][23][24][25], surface conditioned nanoparticles [26][27][28][29][30][31], carbon nanotubes [32][33][34][35], hydrogen absorption/trapping of nanoparticles [36][37][38], nanocrystalline hydroxyapatite [39][40][41][42][43], non-crystalline nanoparticles [44], polymeric nanoparticles [45,46], inorganic-organic nanocomposites [47,48], and doped nanoparticles [49]. Also biomimetic nanostructures are emerging with increased interest [50,51].…”

Sum frequency generation and solid-state NMR study of the structure, orientation, and dynamics of polystyrene-adsorbed peptides

“…In recent years, multidimensional ssNMR experiments incorporating dipolar recoupling pulse sequences have led to high resolution structures of proteins in micro-crystalline form (22,23). Recently, ssNMR techniques have been used to obtain structural parameters such as internuclear distances and bond torsion angles for the biomineralization protein salivary statherin adsorbed onto hydroxyapatite crystal surfaces (24)(25)(26)(27), which combined with the protein structure prediction algorithm RosettaSurface (28) have produced an atomic level structural model (29).…”

“…Recent ssNMR studies of statherin and amelogenin show that proximities of protein side chains to surfaces can be directly detected by rotational echo double resonance (REDOR) recoupling, if the surface contains a NMR-active nucleus such as 31 P or 19 F that is not also ubiquitous in the protein (24,37,38). In the absence of an NMR-active nucleus located exclusively in the surface, protein interactions with charged surfaces can be detected as perturbations of the 13 C chemical shift of certain side chain functional groups, such as carboxyls (27).…”