Probing the Molecular Mechanism of Human Soluble Guanylate Cyclase Activation by NO in vitro and in vivo

Figure 1. Conformational change of FlAsH-labeled sGC β1(1-385)-243TC248, and sGC β1(1-385)-386TC391 upon NO binding (a and b) or CO binding (c and d). The heme concentration was 2 μM in 20 mM HEPES, 150 mM KCl, pH 7.4.

Figure 2. Anisotropy of FlAsH-labeled sGC β1(1-385)-243TC248 and sGC β1(1-385)-386TC391 upon NO binding (a) or CO binding (b). The heme concentration was 3 μM in 20 mM HEPES, 150 mM KCl, pH 7.4. Anisotropy data are the mean ± S.E. of two independent experiments performed in triplicate.

Figure 3. Conformational change of FlAsH-EDT2-labeled sGC β1(1-619)-243TC248 (a) and sGC β1(1-619)-386TC391 (b) upon NO binding. The heme concentration was 3 μM in 20 mM HEPES, 150 mM KCl, pH 7.4.

Figure 4. SRCD spectra in the far-UV region of sGC β1(1-385) (a) and sGC β1(1-385)-243TC248 (b) with and without DEA/NO. The heme concentration of sGC β1(1-385) and sGC β1(1-385)-243TC248 was 50 μM and 45 μM, respectively, in 10 mM HEPES, pH 7.4.

Figure 5. Electronic absorption spectra of sGC α1(1-690)-CFPinsect & β1(1-619)-YFPinsect (a) and sGC CFP-α1(1-690)insect & β1(1-619)-YFPinsect (b) before and after addition of DEA/NO and the mixture of DEA/NO and substrate GTP.

Figure 6. FRET analysis of purified sGC heterodimer. Fluorescent spectra of sGC α1(1-690)-CFPinsect & β1(1-619)-YFPinsect (a) and sGC CFP-α1(1-690) insect & β1(1-619)-YFPinsect (b) at excitation wavelength of 432 nm before and after addition of DEA/NO and the mixture of DEA/NO and substrate GTP.

Figure 7. Co-immunoprecipitation analysis using transiently transfected SH-SY5Y cells expressing the wide type sGC α and β1 subunit or the sGC α and β1 mutant. Cells were co-transfected with HA- and FLAG- tagged constructs, and the HA-tagged constructs were precipitated using anti-HA antibody. Co-precipitated FLAG-tagged constructs were detected by SDS-PAGE/immunoblotting using anti-FLAG antibody (top). As a control, precipitated HA-tagged constructs were detected using anti-HA antibody (bottom). The gels were cropped and the full-length gels were shown in Supplementary Fig. S3.

Figure 8. FLIM-FRET analysis in total cells. The images were taken with SH-SY5Y cells transfected with sGC β1-CFP alone (a), sGC β1-CFP and YFP (b), sGC β1-CFP and sGC β1-YFP (c), sGC β1-CFP and sGC β1-YFP H105A (d), sGC β1-CFP and sGC β1-YFP after addition of DEA/NO (e), sGC β1-CFP and sGC β1-YFP after addition of DEA/NO and GTP (f), and sGC β1-CFP and sGC β1-YFP H105A after addition of DEA/NO (g). Confocal images show the cellular localization of α and β subunits. The rightmost images show the lifetime images. (h) The lifetime values were the average lifetime through fitting the decay curve to the double exponential function from FLIM images. (l) FLIM-FRET efficiency (E) values (mean ± S.E.) were calculated based on CFP lifetime values and obtained over 5–6 cells from 4 to 5 different samples.

Figure 9. FLIM-FRET analysis in total cells. The images were taken with SH-SY5Y cells transfected with sGC α2-CFP alone (a), sGC α2-CFP and YFP (b), sGC α2-CFP and sGC β1-YFP (c), and sGC α2-CFP and sGC β1-YFP after addition of DEA/NO (d). Confocal images show the cellular localization of α and β subunits. The rightmost images show the lifetime images. (e) The lifetime values were the average lifetime through fitting the decay curve to the double exponential function from FLIM images. (f) FLIM-FRET efficiency (E) values (mean ± S.E.) were calculated based on CFP lifetime values and obtained over 5–6 cells from 4 to 5 different samples.

Figure 10. A proposed NO-Induced activation mechanism of sGC.

Figure 11. The domain architectures of sGC and schematic representations of sGC variants constructs used in this study.