Fluorescent Fusion Proteins of Soluble Guanylyl Cyclase Indicate Proximity of the Heme Nitric Oxide Domain and Catalytic Domain 英文参考文献.docVIP

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Fluorescent Fusion Proteins of Soluble Guanylyl Cyclase Indicate Proximity of the Heme Nitric Oxide Domain and Catalytic Domain 英文参考文献.doc

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Fluorescent Fusion Proteins of Soluble Guanylyl Cyclase Indicate Proximity of the Heme Nitric Oxide Domain and Catalytic Domain 英文参考文献

FluorescentFusionProteinsofSolubleGuanylylCyclase IndicateProximityoftheHemeNitricOxideDomainand CatalyticDomain TobiasHaase,NadineHaase,JanRobertKraehling,SoenkeBehrends* Institutfu¨rPharmakologie,ToxikologieundKlinischePharmazie,TechnischeUniversita¨t Braunschweig,Braunschweig,Germany Abstract Background: To examine the structural organisation of heterodimeric soluble guanylyl cyclase (sGC) Fo¨rster resonance energytransfer(FRET)wasmeasuredbetweenfluorescentproteinsfusedtotheamino-andcarboxy-terminalendsofthe sGCb1andasubunits. Methodology/PrincipalFindings:Cyanfluorescentprotein(CFP)wasusedasFRETdonorandyellowfluorescentprotein (YFP)asFRETacceptor.Aftergenerationofrecombinantbaculovirus,fluorescent-taggedsGCsubunitswereco-expressedin Sf9cells.FluorescentvariantsofsGCwereanalyzedinvitroincytosolicfractionsbysensitizedemissionFRET.Co-expression oftheamino-terminallytaggedasubunitswiththecarboxy-terminallytaggedb1subunitresultedinanenzymecomplex thatshowedaFRETefficiencyof10%similartofluorescentproteinsseparatedbyahelixofonly48aminoacids.Because thesefindingsindicatedthattheamino-terminusoftheasubunitsisclosetothecarboxy-terminusoftheb1subunitwe constructedfusionproteinswherebothsubunitsareconnectedbyafluorescentprotein.Theresultingconstructswerenot onlyfluorescent,theyalsoshowedpreservedenzymeactivityandregulationbyNO. Conclusions/Significance: Based on the ability of an amino-terminal fragment of the b1 subunit to inhibit activity of an heterodimerconsistingonlyofthecatalyticdomains(acatbcat),WingerandMarletta(Biochemistry2005,44:4083–90)have proposedadirectinteractionoftheamino-terminalregionofb1withthecatalyticdomains.Insupportofsuchaconceptof ‘‘trans’’regulationofsGCactivitybytheH-NOXdomainsourresultsindicatethatthedomainswithinsGCareorganizedina waythatallowsfordirectinteractionoftheamino-terminalregulatorydomainswiththecarboxy-terminalcatalyticregion. Inaddition,weconstructed‘‘fluorescent-conjoined’’sGC’sbyfusionoftheaamino-terminustotheb1 carbo