Fluorescent fusion proteins of soluble guanylyl cyclase indicate proximity of the heme nitric oxide domain and catalytic domain.

PloS One
Tobias HaaseSoenke Behrends

Abstract

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. 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. Based on the ability of an amino-terminal fragment of the beta1 subunit to inhi...Continue Reading

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Citations

Mar 13, 2012·Annual Review of Biochemistry·Emily R Derbyshire, Michael A Marletta
Feb 12, 2014·Proceedings of the National Academy of Sciences of the United States of America·Melody G CampbellMichael A Marletta
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Jun 3, 2021·International Journal of Molecular Sciences·Elizabeth C Wittenborn, Michael A Marletta

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Methods Mentioned

BETA
FRET

Software Mentioned

BiFC
Per

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