Fluorescence:Atoolforthestudyofmolecularinteractions.(concepts,examples,discussion)Scope:A.Generalconcepts,probes,proteinfluorescenceB.InstrumentalMethods(SSandlifetime)C.Solutequenching(accessibility)D.Anisotropy(rotationalmotion)E.FRET&LRET&Excimers(proximity)Refs:--J.Lakowicz,“PrinciplesofFluorescence Spectroscopy,〞Kluwer,1999 --MolecularProbesHandbook, --MethodsinEnzymology,v.278JABLONSKIDIAGRAMTransitionsbetweenquantizedenergylevelsproducetheabsorptionandfluorescencespectraandvibrationalsubstructure.Consequences:1.Fluorescencespectrumisredshiftedfromabsorption2.Mirrorsymmetryoffluorescenceandabsorption3.ExcitationspectrumshouldmimicabsorptionspectrumJABLONSKIDIAGRAMTheexcitedstatecanbedeactivatedbymanyprocessesafterthesolventrelaxesaroundtheexcitedmolecule.Deactivationprocessesand(rates):1.Fluorescence()2.Solutequenching(kq[Q])3.FRET(kT)4.Everythingelse(ki)5.Stokes’shiftExamplesofFluorophorsFluorescentaminoacidresidues(F,Y,W)(IntrinsicFluorophors)MembraneProbesFluorescentnucleicAcidbase1.Lysreactive;2,Cys-reactive;3,Environmentallysensitive4,pHsensitive;5,Bright;6,Highpolarization(1,3)(2,4,5,6)(1,4,5,6)(1,5,6)(2,3,6)(1,3)ExamplesofSomeFluorescentLabelingReagents

CysReagentsCanuseanasymmetricaldisulfideR1S-SR3(R3=NbS)R1=dansyl,rhodamine,fluorescein,etc.R2=proteinLysReagents(FromMolecularProbesHandbook)ExamplesoflabelingreactionsNon-covalentprobesNote:1.Canobtainbindingstoichiometryandbindingconstant2.InformationabouthydrophobicpocketsGreenFluorescentProtein:Aintrinsicprobewithvisiblefluorescencelex=475nm;lem=515nmAHg-basedCys-specificlabel/probe

(Leavis&Lehrer,1974)ActinTitration(20Xenhancement)RSHg++PSH=RSHgSPSomeFluorescenceApplications

ProteinFluorescenceandEnvironmentL-trp,L-tyr,L-phefluorescenceinwateractinfluorescence:Left:G=native;d=denatured;u=in8MureaRight:decompositionintotrpandtyrcomponents.TrpspectrumisenvironmentallysensitiveActin17tyr,4trp(Lehrer&Kerwar,1972)Lysozyme-(NAG)ninhibitorInteractions(Lehrer&Fasman,1966,1967)InhibitorComplexchangesTrpinactivesiteAcidicquenchinggroupschangeproximitytoTrpBuriedTrpExposedTrpTrpaccessibilityand“solutequenching〞AnisotropyandMotionExcitationwithpolarizedlightproducespolarizedfluorescence.Thedegreeofpolarizationoranisotropydependsonimmobilization.FRETandDistanceMeasurementsFRET=F?rsterResonanceEnergyTransferFRETandassociationreactionsE=1-(DA/

D)orE=1-(FDA/FD)IntensityMeasurementsA=eCl

F≈QI0(1-10-A

)whenA<0.05,F≈QI0AQ=(FAR/FRA)QRI0IFSteady-stateSpectrofluorometerAbsorptionisanabsolutemeasurementFisarelativemeasurement.ARTIFACTSAtA>2,usefrontfaceexcitationExamplesofArtifacts900excitation:filtereffectduetooverlapofemwithex.Frontfaceexcitationfiltereffectduetomorepenetrationat365.A=eCl

F≈QI0(1-10-A

)whenA<0.05,F≈QI0AQ=(FAR/FRA)QRI0IFSteady-stateSpectrofluorometerAbsorptionisanabsolutemeasurementFisarelativemeasurement.LifetimeMeasurementsFrequencyDomainLifetimeMeasurementsTimeDomainWhenI/Io=1/e=0.37,t=.When=45o,=1/=1/2For10nsec,=60MHzTimeDomainvs.FrequencyDomainN-acetyltryptophanamide:anexampleofaprobewithonelifetimeFrequencyDomainSensitivityFrequencyrangeshouldmatchlifetimeLifetimedatagivesmoreinformationthanintensitydataF0=11+22=5When+Q,F=0.5?5+0.5?1=3,Therefore,60%oftotalFisquenched.ForSS,e.g.,intensitychangecouldbeinterpretedas60%quenchingofboth,F=0.5?3+0.5?3=3Butlifetimeshowsthatonly1Trpisquenchedby80%.Onetrpisv.accessible.WithSScaninterpretasallpartlyaccessible.SimilarproblemwithFRET.F0=5F=3CollisionalQuenching1.Collisionalquenchingaffectslifetimeandintensity,staticquenchingonlyaffectsintensity.2.CollisionalquenchingincreaseswithT,staticdecreaseswithT.3.F/Fo=1/(1+KQ[Q]),KQ=Stern-Volmerquenchingconstant,=kq

o,kq=collisionalconstant=ko=1010M-1s-1(=1)CollisionalQuenchingofTrpinProteinsUsingIodideandAcrylamide

SelectivequenchingofTrpfluorescence(intensity)inlysozymebyI-

AcrylamidequenchingofthesingleTrpinLMMmonomer(o)andfilament(?)(Lehrer,BBRC,1967)1.Formultitrpproteinscangetfractionalfluorescenceaccessibility2.Appreciableexposureoftrpregionofmyosinrodinfilament.KQ6.4M-12.8M-1(Zhou,Maeda,Mabuchi,Lehrer,JMB,1998)L-trp(Lehrer,JACS,1970)skatoleLysozyme-triNAGCollisionalQuenchingbyProtonTransferAnisotropy,r,andPolarization,pForrigidrandomprobes,rmax

=0.4,pmax=0.5.rdependsontherelationshipbetweenthelifetimeandthemotion.AnisotropyandRotationalMotion=V/RT=M(v+h)/RTPerrinEquation=lifetime,=rotationalcorrelationtimeExampleofSegmentalMotionR=FB/FFImmobilizationofapeptidebindingtoCaMExampleofImmobilizationofProbeontRNAboundtoRNAsynthetase=V/RTPerrinEquation=lifetime,=rotationalcorrelationtime

Steady-statemeasurementsofAnisotropyAnisotropyDecayMeasurements-TimeDomainr(t)=roexp(-t/)Example:TimeDomainAnisotropyDecayofLADHmeasured=33nscalculated(0.2gH2O/protein)=31nsAnisotropyDecayMeasurement-FrequencyDomain1.Atallfrequencies,theverticalcomponenthasashorterlifetimesoitsprofileisshiftedtohigherfrequencies.2.Atlowfrequencies,plentyoftimeforcompletedepolarization,soI(ll)=I()3..Athighfrequencythephaseshiftismaximumforbothcomponents.IsSSvalueatlowfrequency,roathighfrequency.FrequencyDomainAnisotropyDecayrw=(mll-m)/(mll+2m)SimulatedAnisotropyDecayforSegmentalFlexibilityandProteinRotation(TimeDomain)MobilityofTNSboundtoApoMyoglobinDifferentialPolarizedPhasevs.TimeDomain

UsesofanisotropyMeasurements1.Infoonsizeandshapeofmolecules2.Infoonlocalprobeorsegmentalmotion3.Bindingoflabeledsmallmoleculetobigmolecule.4.Probeofmembranefluidity.5.InformationforFRETBasicsandExamplesofFRETProximityviaFRET,LRET&ExcimersRo,CriticalTransferDistance=1-IDA/ID=1-DA/DExample:FRETbetweenTrpandDansylinHelicalMelittinRo=23.6A(k2=2/3)r=24,4A

DistanceDistributionsAdistancedistributionanalysismightfitbetterthana2oreventhreelifetimeanalysisDistanceDistributionsandUnfolding

LRET“Luminescence(Detected)FoersterResonanceEnergyTransfer〞(LDFRET)

Tb-chelateRh-MalActinTmstructure(Lorenzetal.,1995)LRETAcrossActinFilamentSpectraofCs124StructureofTb-chelateANovelLuminescentDonorCs*+TbTb*+CsNbSSCy-DTPA-Tb-Cs124OverlapBetweenTb+3EmissionandRhodamineAbsorptionChen,Y&Lehrer(2002)TbD*+RhTb+RhA*ms