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1、briefcommunicationsbrightcyanfluorescentSCFP3A,whichareoptimizedforfolding,arethebrightestvari-ants4,5.However,theirquantumyieldsleaveroomforimprove-proteinvariantsment.Sofar,screensforimprovedfluorescentproteinvariantshavebeenaimedatoptimizingthebrightnessofmutantsoftheseidentifiedbyfluo
2、rescenceproteinsbyevaluatingthefluorescenceintensityinindividualbacteriaorcolonies.However,apartfrombrightness,colonylifetimescreeningintensityalsoreflectsexpressionlevel,maturationefficiencyandthicknessofthecolonyandthereforedoesnotnecessarilyreflectthebrightestvariants.JoachimGoedhart1,
3、3,LauravanWeeren1,3,Becauseforagivenfluorophorethequantumyieldandthefluo-MarkAHink1,NorbertOEVischer1,KeesJalink2&rescencelifetimebothincreasewithanincreasedratioofradiative1tononradiativedecayfromtheexcitedstate,wedevisedafluores-TheodorusWJGadellaJrcencelifetime–basedscreentooptimizequa
4、ntumyield,therebyimprovingtheintrinsicmolecularbrightness.Amajoradvantageoptimizationofautofluorescentproteinsbyintensity-basedisthatthefluorescencelifetimecanbedeterminedindependentlyscreeningofbacteriadoesnotnecessarilyidentifytheoffluorescenceintensity.Fromsuchascreen,fluorescentprotei
5、nsbrightestvariantforeukaryotes.Wereportastrategytoscreencanalsobeselectedforsingle-exponentialdecay,whichisimpor-excitedstatelifetimes,whichidentifiedcyanfluorescenttantfortime-resolvedFRETmeasurements.proteinswithlongfluorescencelifetimes(>3.7ns)andOurscreeningsetup(SupplementaryFig.1)w
6、asbasedonahighquantumyields(>0.8).onevariant,mturquoise,wasfrequencydomainfluorescencelifetimeimagingmicroscope6,1.5-foldbrighterthanmceruleaninmammaliancellsenablingacquisitionofafluorescencelifetimeimageofanentireanddecayedmono-exponentially,makingitanexcellentPetridishwithinsecondsfrom
7、whichthefluorescencelifetime(andfluorescenceresonanceenergytransfer(fret)donor.intensity)ofindividualbacterialcoloniescanbedetermined.Cyanfluorescentproteinvariants1derivedfromAequoreavictoriaGFParewidelyab4.0mTurquoiseappliedforlocalizationandfluorescencemCeruleanY
