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MOLECULAR RECOGNITION AT THE PROTEIN-HYDROXYAPATITE INTERFACE
Patrick S. Stayton1,*,
Gary P. Drobny2,*,
Wendy J. Shaw2,
Joanna R. Long1 and
Michele Gilbert1
1 Departments of Bioengineering, Box 351721, and
2 Chemistry, Box 351700, University of Washington, Seattle, WA 98195;
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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.
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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.
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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.
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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.
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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.
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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.
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Critical Reviews in Oral Biology & Medicine, Vol. 14, No. 5,
370-376 (2003)
DOI: 10.1177/154411130301400507
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-helix and 55% β-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