fluorescent fusion proteins of soluble guanylyl cyclase indicate proximity of the heme nitric oxide domain and catalytic domain荧光融合蛋白的可溶性guanylyl环化酶显示邻近的血红素一氧化氮域和催化领域.pdfVIP

Fluorescent Fusion Proteins of Soluble Guanylyl Cyclase Indicate Proximity of the Heme Nitric Oxide Domain and Catalytic Domain Tobias Haase, Nadine Haase, Jan Robert Kraehling, Soenke Behrends* ¨ ¨ Institut fur Pharmakologie, Toxikologie und Klinische Pharmazie, Technische Universitat Braunschweig, Braunschweig, Germany Abstract ¨ Background: To examine the structural organisation of heterodimeric soluble guanylyl cyclase (sGC) Forster resonance energy transfer (FRET) was measured between fluorescent proteins fused to the amino- and carboxy-terminal ends of the sGC b1 and a subunits. Methodology/Principal Findings: Cyan fluorescent protein (CFP) was used as FRET donor and yellow fluorescent protein (YFP) as FRET acceptor. After generation of recombinant baculovirus, fluorescent-tagged sGC subunits were co-expressed in Sf9 cells. Fluorescent variants of sGC were analyzed in vitro in cytosolic fractions by sensitized emission FRET. Co-expression of the amino-terminally tagged a subunits with the carboxy-terminally tagged b1 subunit resulted in an enzyme complex that showed a FRET efficiency of 10% similar to fluorescent proteins separated by a helix of only 48 amino acids. Because these findings indicated that the amino-terminus of the a subunits is close to the carboxy-terminus of the b1 subunit we constructed fusion proteins where both subunits are connected by a fluorescent protein. The resulting constructs were not only fluorescent, they also showed preserved enzyme activity and regulation by NO. Conclusions/Significance: Based on the ability of an amino-terminal fragment of the b1 subunit to inhibit activity of an heterodimer consisting