Institut für Pharmakologie, Toxikologie und Klinische Pharmazie, Technische Universität Braunschweig, Braunschweig, Germany.
PLoS One. 2010 Jul 15;5(7):e11617. doi: 10.1371/journal.pone.0011617.
To examine the structural organisation of heterodimeric soluble guanylyl cyclase (sGC) Förster resonance energy transfer (FRET) was measured between fluorescent proteins fused to the amino- and carboxy-terminal ends of the sGC beta1 and alpha 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 alpha subunits with the carboxy-terminally tagged beta1 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 alpha subunits is close to the carboxy-terminus of the beta1 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 beta1 subunit to inhibit activity of an heterodimer consisting only of the catalytic domains (alphacatbetacat), Winger and Marletta (Biochemistry 2005, 44:4083-90) have proposed a direct interaction of the amino-terminal region of beta1 with the catalytic domains. In support of such a concept of "trans" regulation of sGC activity by the H-NOX domains our results indicate that the domains within sGC are organized in a way that allows for direct interaction of the amino-terminal regulatory domains with the carboxy-terminal catalytic region. In addition, we constructed "fluorescent-conjoined" sGC's by fusion of the alpha amino-terminus to the beta1 carboxy-terminus leading to a monomeric, fluorescent and functional enzyme complex. To our knowledge this represents the first example where a fluorescent protein links two different subunits of a higher ordered complex to yield a stoichometrically fixed functionally active monomer.
为了研究异二聚体可溶性鸟苷酸环化酶（sGC）的结构组织，我们测量了融合到 sGCβ1 和α亚基氨基末端和羧基末端的荧光蛋白之间的Förster 共振能量转移（FRET）。
方法/主要发现：青色荧光蛋白（CFP）用作 FRET 供体，黄色荧光蛋白（YFP）用作 FRET 受体。生成重组杆状病毒后，在 Sf9 细胞中共同表达荧光标记的 sGC 亚基。通过敏化发射 FRET 在细胞质部分体外分析荧光 sGC 变体。氨基末端标记的α亚基与羧基末端标记的β1 亚基的共表达导致酶复合物的 FRET 效率为 10%，类似于仅由 48 个氨基酸组成的螺旋隔开的荧光蛋白。由于这些发现表明α亚基的氨基末端靠近β1 亚基的羧基末端，我们构建了融合蛋白，其中两个亚基通过荧光蛋白连接。所得构建体不仅具有荧光，而且还显示出保留的酶活性和对 NO 的调节。
结论/意义：基于β1 亚基氨基末端片段抑制仅由催化结构域（αcatβcat）组成的异二聚体活性的能力，Winger 和 Marletta（Biochemistry 2005，44：4083-90）提出了β1 的氨基末端区域与催化结构域的直接相互作用。为了支持 sGC 活性的“反式”调节的这种概念，我们的结果表明，sGC 中的结构域以允许氨基末端调节结构域与羧基末端催化区域直接相互作用的方式组织。此外，我们通过将α氨基末端融合到β1 羧基末端来构建“荧光连接”的 sGC，导致单体、荧光和功能性酶复合物。据我们所知，这代表了第一个荧光蛋白将两个不同的亚基连接到更高阶复合物以产生化学计量固定的功能性单体的例子。
