Fig 1.
Schematic illustration of the proteoliposomes and structure of integrin αIIbβ3.
A) Schematic illustration of the proteoliposomes as obtained after the reconstitution procedure before adsorption on SiO2 surface. αIIbβ3 (αIIb-subunit in blue and β3-subunit in orange) is reconstituted into liposomes and treated with Triton X-100 as well as biobeads and activated by manganese ions (Mn2+) or drugs. B) Structure of αIIbβ3 in bent (left) and open/active (right) conformation in a DMPG:DMPC (1:20) lipid membrane (cyan). The integrin model combines the αIIbβ3 transmembrane domain (PDB-code 2k9j) and ectodomain (PDB-code 3fcs), missing residues were added as random coils. The VMD 1.9. and PyMOL 2.1. softwarepackages were used to create this figure.
Fig 2.
Validation of αIIbβ3 reconstitution into liposomes.
A) TEM images of proteoliposomes. The inset shows a close-up view of αIIbβ3 (indicated by arrow) incorporated in a membrane environment. B) DLS data showing the hydrodynamic diameter of liposomes (black) and proteoliposomes (red) of three independent experiments measured in liposome buffer at 37°C. C) Statistical analysis of FACS-plots with liposomes and proteoliposomes from three independent measurements. Percentages of the mean ± standard error of the mean (SEM) of anti-CD41 (red) and anti-CD61 binding (blue) on PE CF- liposomes were plotted. D) Reductive SDS-PAGE of liposomes (-) and proteoliposomes (+), protein molecular weight standard (M) is shown on the left. The bands corresponding to the αIIb- (red) and β3-subunit (blue) are indicated by arrows.
Fig 3.
A) Activation assay using PAC-1 antibody. Each value is the mean of three replicate measurements ± SEM. The binding of PE CF-liposomes/proteoliposomes to 5 μg/mL PAC-1 coated on a microtiter plate was detected after incubation with buffer (green), 1 mM Mn2+ (red) and 5 mM EDTA (blue). Values of bare liposome samples were subtracted from proteoliposome values. The y-axis shows relative fluorescence units (RFU). B) Integrin activation investigated by flow cytometry with PE CF-liposomes (black)/proteoliposomes (red) by adding PAC-1-Alexa 647-coupled antibody. Percentages of the mean ± SEM of Alexa-647 signal of PE positive events incubated with buffer, Mn2+ and EDTA are shown. C) Representative QCM-D data showing the changes in frequency f (top) and dissipation D (bottom) of the seventh overtone for the binding of the conformation-specific antibody PAC-1 at 37°C. Buffer was injected over the SiO2 sensors (phase I) and after reaching a baseline liposomes or proteoliposomes were injected and the formation of a bilayer was observed (phase II). After a washing step with either liposome buffer, liposome buffer with 1 mM Mn2+, or 5 mM EDTA (phase III), PAC-1 antibody was injected (phase IV) and binding was observed (indicated by arrows). Rinsing with the respective buffer followed (phase V).
Fig 4.
A) Far-UV region CD spectra of αIIbβ3 reconstituted into liposomes in buffer (green), with addition of 1 mM Mn2+ (red) or 5 mM EDTA (blue). One representative spectrum recorded with proteoliposomes with a protein concentration of approximately 0.4 μM or liposomes in 5 mm path length cuvettes at 37°C is shown. Liposome spectra were subtracted from the respective proteoliposome spectra. B) MDS demonstrating the regions that changed drastically in the antiparallel β-sheet probability after removing all structural ions (right) or converting the three Ca2+ in the MIDAS, ADMIDAS and SyMBS to Mn2+ (left). The red and blue colored regions illustrate the formation or loss of β-sheets, respectively, compared to integrin with Ca2+ and Mg2+ metal ions only. In white or transparent regions, the β-sheet probability did not change.
Fig 5.
A) Changes in frequency (Δf) and dissipation (ΔD) after PAC-1 injection event (indicated by the respective arrows) in representative QCM-D experiments with proteoliposomes after treatment with buffer (green), 1 mM Mn2+ (red), 250 μg/mL fondaparinux (purple), 50 μg/mL quinine (black), 250 μg/mL UFH (blue) and 5 mM EDTA (light blue) for at least 15 min at 37°C. B) Drug activation assay using PAC-1 antibody. Each value is the mean of three replicate measurements ± SEM. The binding of PE CF-liposomes/proteoliposomes to 5 μg/mL PAC-1 coated to a microtiter plate was detected after the incubation with buffer (green), 1 mM Mn2+ (red), 250 μg/mL fondaparinux (purple), 250 μg/mL UFH (blue) and 50 μg/mL quinine (black). Liposome sample results were subtracted from proteoliposomes values. The y-axis shows relative fluorescence units (RFU). C) Normalized single wavelength plot for corresponding MRDE values at 210 nm from far-UV region CD spectra of αIIbβ3 incorporated into liposomes/proteoliposomes treated with increasing concentrations of UFH (blue), fondaparinux (purple) and quinine (black), respectively. Normalized averages from three independent measurements ± SEM are shown as dots that were recorded with proteoliposomes with a protein concentration of approximately 0.4 μM or liposomes in 5 mm path length cuvettes at 37°C. Liposomes spectra were subtracted from the respective proteoliposome spectra. Respective trendlines were applied to guide the reader.