Sensitive Detection of p65 Homodimers Using Red-Shifted and Fluorescent Protein-Based FRET Couples 英文参考文献.docVIP

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Sensitive Detection of p65 Homodimers Using Red-Shifted and Fluorescent Protein-Based FRET Couples 英文参考文献.doc

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Sensitive Detection of p65 Homodimers Using Red-Shifted and Fluorescent Protein-Based FRET Couples 英文参考文献

SensitiveDetectionofp65HomodimersUsingRed- ShiftedandFluorescentProtein-BasedFRETCouples JoachimGoedhart*,JoopE.M.Vermeer,MerelJ.W.Adjobo-Hermans,LauravanWeeren,TheodorusW.J.GadellaJr. SectionofMolecularCytology,CentreforAdvancedMicroscopy,SwammerdamInstituteforLifeSciences,UniversityofAmsterdam,Amsterdam,The Netherlands Background.FluorescenceResonanceEnergyTransfer(FRET)betweenthegreenfluorescentprotein(GFP)variantsCFPand YFP is widely used for the detection of protein-protein interactions. Nowadays, several monomeric red-shifted fluorescent proteinsareavailablethatpotentiallyimprovetheefficiencyofFRET.Methodology/PrincipalFindings.Toallowside-by-side comparison of several fluorescent protein combinations for detection of FRET, yellow or orange fluorescent proteins were directlyfusedtoredfluorescentproteins.FRETfromyellowfluorescentproteinstoredfluorescentproteinswasdetectedby both FLIM and donor dequenching upon acceptor photobleaching, showing that mCherry and mStrawberry were more efficientacceptorsthanmRFP1.Circularpermutatedyellowfluorescentproteinvariantsrevealedthatinthetandemconstructs the orientation of the transition dipole moment influences the FRET efficiency. In addition, it was demonstrated that the orangefluorescentproteinsmKOandmOrangearebothsuitableasdonorforFRETstudies.Themostfavorableorange-red FRETpairwasmKO-mCherry,whichwasusedtodetecthomodimerizationoftheNF-kBsubunitp65insinglelivingcells,with athreefoldhigherlifetimecontrastandatwofoldhigherFRETefficiencythanforCFP-YFP.Conclusions/Significance.The observed high FRET efficiency of red-shifted couples is in accordance with increased Fo¨rster radii of up to 64 A,? being significantlyhigherthantheFo¨rsterradiusofthecommonlyusedCFP-YFPpair.Thus,red-shiftedFRETpairsarepreferablefor detectingprotein-proteininteractionsbydonor-basedFRETmethodsinsinglelivingcells. Citation: GoedhartJ,VermeerJEM,Adjobo-HermansMJW,vanWeerenL,GadellaTWJJr(2007)SensitiveDetectionofp65HomodimersUsingRed- ShiftedandFluo