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MOLECULAR RECOGNITION AT THE PROTEIN-HYDROXYAPATITE INTERFACE

¹ Departments of Bioengineering, Box 351721, and ² Chemistry, Box 351700, University of Washington, Seattle, WA 98195;

View larger version (22K): [in a new window]   Figure 1. Strategies for determining statherin structure on HAP and summary results for the N-terminal domain. The sequence of statherin is given, along with the 13C-labeled amino acid positions. DRAWS and DQDRAWS determine the backbone torsional angle phi at the pSpS position, while REDOR was used to determine the (i) to (i+4) 13C-15N hydrogen bonding distances between S3 and F7, and L8 and G12. Peptides were synthesized by standard Fmoc solid-phase peptide synthesis strategies, and phosphorylation was performed after protein synthesis for serine 13C=O enriched samples. The HAP crystals had a surface area of 80 m2/g, and adsorption of statherin was carried out at 40 µM for two hours, followed by extensive washing in PBS to remove loosely adsorbed protein.

View larger version (16K): [in a new window]   Figure 2. DQDRAWS and REDOR characterization of statherin structure on HAP. The left panel displays the results of the DQDRAWS experiment to measure the backbone angle phi at the pS-pS position. The filled diamonds are data on the hydrated sample, the open diamonds are for the same sample that was subsequently lyophilized, and the dotted line is a simulated data fit to 45% -helix and 55% ß-sheet. The data were taken at room temperature on a 500-MHz home-built spectrometer with a 4-kHz spinning speed, a 34-kHz 13C RF field, and 100-kHz proton decoupling. The REDOR experiment shown in the right panel was performed on a Chemagnetics Infinity 300 spectrometer at a spinning speed of 4 kHz, a 43-kHz 13C RF field, a 45-kHz 15N RF field, XY8 phase cycling on both channels, and 70-kHz decoupling. Measurements on the hydrated samples were done at -25°C to remove any motions on the timescale of the REDOR experiment (as verified by T1 relaxation measurements). The solid lines represent fits to distances of 4.2 Å for the hydrated sample and 5.2 Å for the sample that was lyophilized after adsorption in buffered solution. The dotted lines are fits to 5.2 Å and 45% -helix, 55% ß-sheet.

View larger version (30K): [in a new window]   Figure 3. Dynamic studies of the statherin N15 peptide on HAP. 13C CPMAS spectra of N15 peptides that were adsorbed to HAP crystals of 77 m2/g surface area from 2-mM peptide solutions in modified PBS (100 mM NaCl, 40 mM KCl, 4.3 mM Na2HPO4, and 1.4 mM KH2PO4). After the hydrated sample data were collected, the same samples were frozen and lyophilized in the rotor. Each spectrum consists of 10,000 scans taken on a spectrometer operating at a 125.74-MHz 13C frequency with a 3-kHz sample spinning rate.

View larger version (20K): [in a new window]   Figure 4. Dynamic studies of statherin on HAP crystals. CPMAS spectra were acquired at room temperature on a home-built 500-MHz spectrometer with spinning speeds of 3, 4, and 5 kHz. Cross-polarization was accomplished with a 2-msec contact time at RF fields of 50 kHz, and data were collected during 100-kHz proton decoupling. T1 measurements were done at a spinning speed of 6 kHz on a Chemagnetics Infinity 300 with 13C spin lock times of 0.05 to 4.55 msec at an RF field of 42 kHz after 1.5 msec of cross-polarization with 70-kHz proton decoupling during acquisition.

View larger version (39K): [in a new window]   Figure 5. Schematic design of N15 fusion peptides to display integrin-binding sequences. The functional DGEA and RGD sequences from collagen 1 and osteopontin, respectively, were fused to N15 with a proline linker. Subsequent signal pathway studies have shown that only the DGEA fusion peptide activates ERK1/2 phosphorylation, while both peptides direct FAK phosphorylation.

View larger version (31K): [in a new window]   Figure 6. Solid-state NMR characterization of the RGD domain dynamics of the N15 fusion peptide on HAP. 13C CPMAS spectra were collected on a Chemagnetics CMX Infinity spectrometer operating at a 13C frequency of 125.72 MHz using a Chemagnetics doubly-resonant MAS probe. These experiments used a 1H 90° pulsewidth of 7.5 msec followed by a contact time of 1.5 msec and a spinning speed of 3003 Hz. The surface-adsorbed samples were signal-averaged for 10,240 scans. The C-terminal glycine retained the large-amplitude dynamics typical of the N15 C-terminus, suggesting that the aspartic acid is not strongly interacting with the HAP surface. The RGD domain dynamics at this position may underlie the excellent cell-binding activity of this peptide when immobilized on HAP surfaces.

View larger version (17K): [in a new window]   Figure 7. Characterization of MC3T3-E1 cell adhesion to HAP-immobilized N15-fusion peptides. Both the N15-DGEA and N15-RGD peptides bind the MC3T3-E1 cells in a dose-dependent fashion, while control N15-DGAA and N15-RGE peptides do not bind cells above background control levels. Antibody-blocking studies demonstrated that immobilized N15-PGRGDS bound MC3T3-E1 osteoblasts predominantly via the vß3 integrin, and immobilized N15-PDGEA bound MC3T3 E1 osteoblasts predominantly through the 2ß1 integrin.

View larger version (16K): [in a new window]   Figure 8. Molecular model of the statherin N-terminal domain. The positions used in the REDOR characterization of Fig. 2 are colored. The first red residue is Ser 3, and its corresponding REDOR partner label is Phe 7, colored blue. The second distance was measured from the red position at Leu 8 to the blue position at Gly 12.