Figure 4
Figure 4. Binding of heme to C3. SPR analysis of the binding of different concentrations of heme to (A) AP proteins and (B) C3 fragments immobilized on the chip (mean ± SD from 3 experiments on 2 chips). The flow rate was 30 μL/min with N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) running buffer (10 mM HEPES, 150 mM NaCl, Tween 0.005%; pH 7.3). Each interaction was tested on at least two different chips at least twice per chip. Data were double referenced and analyzed by using ProteOn manager software. (C) Absorption spectroscopy for the binding of heme at a 10-fold molar excess to C3, C3b, and C3a. Dashed gray line represents the spectrum of heme in PBS and the black line represents the spectrum of heme in the presence of protein. (D) Dose-dependence of the binding of heme to C3, C3b, and C3a. Heme was added to the protein solution and incubated for 2 minutes in the dark at 20°C before recording the spectrum. Absorbance differences between protein-bound hemin and hemin at absorbance maxima (λ = 395 nm) in the Soret region (ΔA) were used to build titration binding curves. (E) Molecular docking of heme to C3: heme (red spheres) in all 14 retrieved models docked to three regions of the molecule (I, II, and III), one being close to the ANA domain (cyan), the second being close to the thioester bond (green) in the TED domain (blue), and the third again being close to the TED domain. (F) Zoomed in view of the II cluster with the top-score heme molecule visualized using ball-and-stick art in red. The distance between the thioester bond (green) and heme is less than 10 Å. (G) SPR analysis of the binding of heme-exposed C3 to C3 immobilized on the chip. One experiment of 3 with similar results is presented. No interaction was detected in the absence of heme, although dose-dependent binding was found with increasing heme concentrations. (H) Size exclusion chromatography of native C3 and heme-exposed C3. Fast protein liquid chromatography with a Superose 6 column with dual wavelength was used (280 nm for C3, black line; 400 nm for heme, gray dashed line). C3 at 6.4 µM was incubated for 10 minutes on ice with PBS or 64 µM heme, giving 10-fold molar excess adjusted to a final volume of 1 mL and loaded to the column.

Binding of heme to C3. SPR analysis of the binding of different concentrations of heme to (A) AP proteins and (B) C3 fragments immobilized on the chip (mean ± SD from 3 experiments on 2 chips). The flow rate was 30 μL/min with N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) running buffer (10 mM HEPES, 150 mM NaCl, Tween 0.005%; pH 7.3). Each interaction was tested on at least two different chips at least twice per chip. Data were double referenced and analyzed by using ProteOn manager software. (C) Absorption spectroscopy for the binding of heme at a 10-fold molar excess to C3, C3b, and C3a. Dashed gray line represents the spectrum of heme in PBS and the black line represents the spectrum of heme in the presence of protein. (D) Dose-dependence of the binding of heme to C3, C3b, and C3a. Heme was added to the protein solution and incubated for 2 minutes in the dark at 20°C before recording the spectrum. Absorbance differences between protein-bound hemin and hemin at absorbance maxima (λ = 395 nm) in the Soret region (ΔA) were used to build titration binding curves. (E) Molecular docking of heme to C3: heme (red spheres) in all 14 retrieved models docked to three regions of the molecule (I, II, and III), one being close to the ANA domain (cyan), the second being close to the thioester bond (green) in the TED domain (blue), and the third again being close to the TED domain. (F) Zoomed in view of the II cluster with the top-score heme molecule visualized using ball-and-stick art in red. The distance between the thioester bond (green) and heme is less than 10 Å. (G) SPR analysis of the binding of heme-exposed C3 to C3 immobilized on the chip. One experiment of 3 with similar results is presented. No interaction was detected in the absence of heme, although dose-dependent binding was found with increasing heme concentrations. (H) Size exclusion chromatography of native C3 and heme-exposed C3. Fast protein liquid chromatography with a Superose 6 column with dual wavelength was used (280 nm for C3, black line; 400 nm for heme, gray dashed line). C3 at 6.4 µM was incubated for 10 minutes on ice with PBS or 64 µM heme, giving 10-fold molar excess adjusted to a final volume of 1 mL and loaded to the column.

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