期刊
PLOS ONE
卷 5, 期 7, 页码 -出版社
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0011617
关键词
-
资金
- VolkswagenStiftung
- Deutsche Forschungsgemeinschaft [INST 188/286-1]
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 beta(1) 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 beta(1) 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 beta(1) 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 beta(1) subunit to inhibit activity of an heterodimer consisting only of the catalytic domains (alpha(cat)beta(cat)), Winger and Marletta (Biochemistry 2005, 44: 4083-90) have proposed a direct interaction of the amino-terminal region of beta(1) 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 beta(1) 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.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据