Fluorescent fusion proteins of soluble guanylyl cyclase indicate proximity of the heme nitric oxide domain and catalytic domain.
BACKGROUND: 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: Cya...
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2010-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC2904703?pdf=render |
| _version_ | 1831773366148661248 |
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| author | Tobias Haase Nadine Haase Jan Robert Kraehling Soenke Behrends |
| author_facet | Tobias Haase Nadine Haase Jan Robert Kraehling Soenke Behrends |
| author_sort | Tobias Haase |
| collection | DOAJ |
| description | BACKGROUND: 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. |
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| id | doaj.art-1b7878779614400cb7d6f0ccd113b5cf |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-22T08:35:05Z |
| publishDate | 2010-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-1b7878779614400cb7d6f0ccd113b5cf2022-12-21T18:32:23ZengPublic Library of Science (PLoS)PLoS ONE1932-62032010-01-0157e1161710.1371/journal.pone.0011617Fluorescent fusion proteins of soluble guanylyl cyclase indicate proximity of the heme nitric oxide domain and catalytic domain.Tobias HaaseNadine HaaseJan Robert KraehlingSoenke BehrendsBACKGROUND: 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.http://europepmc.org/articles/PMC2904703?pdf=render |
| spellingShingle | Tobias Haase Nadine Haase Jan Robert Kraehling Soenke Behrends Fluorescent fusion proteins of soluble guanylyl cyclase indicate proximity of the heme nitric oxide domain and catalytic domain. PLoS ONE |
| title | Fluorescent fusion proteins of soluble guanylyl cyclase indicate proximity of the heme nitric oxide domain and catalytic domain. |
| title_full | Fluorescent fusion proteins of soluble guanylyl cyclase indicate proximity of the heme nitric oxide domain and catalytic domain. |
| title_fullStr | Fluorescent fusion proteins of soluble guanylyl cyclase indicate proximity of the heme nitric oxide domain and catalytic domain. |
| title_full_unstemmed | Fluorescent fusion proteins of soluble guanylyl cyclase indicate proximity of the heme nitric oxide domain and catalytic domain. |
| title_short | Fluorescent fusion proteins of soluble guanylyl cyclase indicate proximity of the heme nitric oxide domain and catalytic domain. |
| title_sort | fluorescent fusion proteins of soluble guanylyl cyclase indicate proximity of the heme nitric oxide domain and catalytic domain |
| url | http://europepmc.org/articles/PMC2904703?pdf=render |
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