| Endogenously NO is a multifunctional physiological messenger that has been implicated in the regulation of vasodilatation, smooth muscle relaxation and platelet aggregation in the cardiovascular system,while in the central nervous system (CNS) it involved in neural development, synaptic transmission, synaptic plasticity and formation of long-term memory (LTM). Soluble guanylate cyclase (sGC) is a major receptor for NO-signaling, catalyzing guanosine-5’-triphosphate (GTP) to cyclic guanosine-3’,5’-monophosphate (cGMP)。 Dysfunction of NO signaling results in many pathological disorders, ranging from several cardiovascular diseases, such as arterial hypertension, pulmonary hypertension, heart failure, atherosclerosis and restenosis, to neurodegenerative diseases. Therefore, research on sGC is very important to reveal the pathological mechanism of significant diseases, and provide ideas for drug design.sGC in eukaryote is a heterodimeric hemoprotein, composed of a a-subunit and a β-subunit. For human sGC, there are two isoforms in vivo α1β1and α1β1. The biggest difference between these two isoforms lies in their cellular localization and tissue distribution. The α1β1is found in the cytosolic fractions of vascularized tissues,while the α2β1is major in brain、placenta and uterus. α2β1can interact with synaptic adaptor protein postsynaptic density-95(PSD-95). So it is likely that α2β1has an important role in the mediation of interneuronal communication in brain, which is involved in learning and memory formation. Since most of studies of sGC were focus on α1β1, few knowledge about α2β1is known, especially on molecular level.In this study, we constructed plasmids for N-terminal heme domain and intermediate domains of hsGC α2β1, C-terminal catalytic domain and full-length of hsGC a2. We developed efficient expression and purification methods for heme domain protein (hsGC α2H), heterodimeric heme domain protein (hsGC β1H-α2H, hsGC α2H-β1H) and heterodimeric coiled coil domain protein (hsGC α2cc-β1cc)-The purified protein was characterized by SDS-PAGE, gel filtration chromatography, and matrix assisted laser desorption ionization/time of flight MS (MALDI-TOF-MS).Structural characterization and properties study are focus on heme domain of hsGC α2β1Through pyridine hemochromogen method and Bradford’s protein assay, we confirmed that95%of reconstituted hsGC α2H, hsGC β1H-α2H, hsGC α2H-β1H are holo-proteins with heine. The electronic absorption spectra of three proteins in different forms are measured. The electronic paramagnetic spectroscopy (EPR) was applied to characterize the mental center of hsGC α2H、hsGC β1H-α2H and hsGC α2H-β1H.The EPR spectra of the three ferric protein indicated that the axial positions of heme are vacant or occupied by weak ligands, such as H2O. The NO complexes in reduced state exhibited g values of gx=2.009, gz=2.068with triplet hyperfine splitting of NO, characteristic of five-coordinated heme-nitrosyl species, which are similar to hsGC β195, hsGC a259and as-isolated heterodimer sGC. So we concluded that the three protein were in their functional states.CO binding to hsGC a2H, hsGC β1H-a2H, hsGC α2H-β1H by CO titration was monitored spectroscopically. There is an isosbestic point at428nm for hsGC α2H and hsGC β1H-α2H, indicating that CO-bound were formed by one-step binding without any intermediate product during the CO binding process. In the case of hsGC α2H-β1H, no isosbestic point was observed, suggesting that at least one intermediate product was formed accompanied with CO binding. The measured dissociation constant (Kd) for hsGC α2H and hsGC β1H-α2H is2.2μM and1.6μM, respectively, while for hsGC α2H-β1H is1.1μM. Dissociation of NO from the heme of hsGC was estimated using the CO/dithionite trapping method. The plots of NO dissociation from hsGC α2H and hsGC β1H-α2H were best fitted with a double exponential equation. This result suggested that the NO dissociation from hsGC a2H and hsGC β1H-α2H were complex with at least two release phases. It was proposed that the NO-bound hsGC α2H and hsGC β1H-α2H are a mixture of two different5-coordinate species with open and closed conformation.Heme dissociation rate constants (koff) were measured by UV-vis spectrometer with kinetic mode from the heme transfer experiments using the apo-myoglobin assay. We found that the heme dissociation of sGC is much faster than that of other hemoproteins, and this might be the possible reason for heme to be lost during purification. In addition, we study secondary structure of hsGC α2β1heme domain by circular dichroism spectroscopy and Fourier transform infrared spectroscopy. We found that reconstituted heme disturber the secondary structure of hsGC α2H-β1H.In summary, we established an efficient expression and purification system for hsGC α2β1, which is the foundation of structure and mechanism. All these results are beneficial for understanding of the overall structure of heme binding site of hsGC α2β11and NO signaling mechanism. |
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