Introduction of fluorescence protien.docx
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Introduction of fluorescence protien.docx
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Introductionoffluorescenceprotien
Introduction
Althoughthefirstreportoffluorescenceemissioninthebioluminescenthydrozoanjellyfishspecies Aequoreavictoria wasrecordedinthemid-twentiethcenturyandaproteinextractwasindependentlydemonstratedbytwoinvestigatorstoberesponsibleforthis"green"fluorescenceinthe1960sand1970s,ittookanother20yearsandanumberofsignificantadvancesinthetechnologyofmolecularandcellularbiologytowitnesstheelucidationoftheprimaryaminoacidstructure.Followingshortlythereafterwastheastonishingdemonstrationthatthejellyfishgreenfluorescentprotein(GFP;Figure1)couldbereadilyemployedasausefulmarkerforgeneexpressionincellsevolutionarilyfarremovedfromthejellyfish.
Duringthenextseveralyears,anumberof"enhanced"geneticvariantsfeaturingfluorescenceemissionspectralprofilesspanningtheblue,cyan,green,andyellowregionsofthevisiblespectrumweredevelopedbyengineeringspecificmutationsintotheoriginalGFPnucleotidesequence.Oneofthemostsignificantadvancesfollowingtheinitialcloningandearlymutagenesiseffortsonthe Aequoreavictoria greenfluorescentproteinwasthediscoveryofsimilarproteinsinnon-bioluminescentreefcoralsandseaanemones,whichnotonlyprovidedalargespectrumofnewemissioncolors,butalsodemonstratedthattheseproteinmotifsoccurinawiderangeoforganisms.
Overthepastdecade,fluorescentproteinshavelaunchedanewandunprecedentederaincellbiologybyenablinginvestigatorstoapplyroutinemolecularcloningmethods,fusingtheseopticalprobestoawidevarietyofproteinandenzymetargets,inordertomonitorcellularprocessesinlivingsystemsusingfluorescencemicroscopyandrelatedmethodology.Thespectrumofapplicationsforfluorescentproteinsrangesfromreportersoftranscriptionalregulationandtargetedmarkersfororganellesandothersubcellularstructurestofusionproteinsdesignedtomonitormotilityanddynamics.Thesefascinatingprobeshavealsoopenedthedoortocreatingbiosensorsfornumerousintracellularphenomena,includingpHandionconcentrationfluctuations,proteinkinaseactivity,apoptosis,voltage,andcyclicnucleotidesignaling.Byapplyingselectedpromotersandtargetingsignals,fluorescentproteinbiosensorscanbeintroducedintoanintactorganismanddirectedtoahostofspecifictissues,celltypes,aswellassubcellularcompartmentstoenableanunprecedentedfocusonmonitoringavarietyofphysiologicalprocesses.
Whencoupledtorecenttechnicaladvancesinwidefieldfluorescenceandconfocalmicroscopy,includingultra-fastlowlightleveldigitalCCDcameras,spinningdisk,structuredillumination,andswept-fieldinstruments,aswellasmultitrackinglaserscanningsystemswithacousto-optictunablefilter(AOTF)control,thegreenfluorescentproteinanditscolor-shiftedgeneticderivativeshavedemonstratedinvaluableserviceinmanythousandsoflive-cellimagingexperiments.Comparedtomanytraditionalsyntheticfluorophores,whichareoftentoxicorphotoreactive,theuseoffluorescentproteinsisminimallyinvasiveforlivingcells,enablingvisualizationandrecordingoftime-lapseimagesequencesforextendedperiodsoftime.Furthermore,continuedadvancesingeneticallyfine-tuningthepropertiesoffluorescentproteinvariantshaveledtoincreasedbrightnesslevels,improvedphotostability,andsignificantlybetterexpressioninmammaliancells.Thesefunctionalenhancementshavestimulatedawidevarietyofinvestigationsintoproteindynamicsandfunctionusingfluorescentproteinchimerasimagedatlowlightintensitiesformanyhourstoextractvaluableinformationaboutchangesinthesteady-statedistribution.
IllustratedinFigure2isan Aequoreavictoria GFPmutationmapshowingmanyofthecommonmutationssuperimposedonatopologicallayoutofthepeptidestructure.The beta-sheetsarenumberedanddepictedasthin,greencylinderswithanarrowpointingtowardstheC-terminus,whereas alpha-helicesaredepictedbybluebarrels.Mutationsarecolor-codedtorepresentthevariantstowhichtheyapply:
BFPs(blue),CFPs(cyan),GFPs(green),YFPs(yellow),andSapphire(violet).Folding,shared,andmonomerizingmutationsareindicatedwithgrayellipses.Notethatalmost75percentofthemutationsarelocatedinthecentralhelixand beta-sheetstrands7,8,and10.Ingeneral,wavelength-specificmutationsoccurnearthecentralhelixcontainingthechromophore,whilefoldingmutationsoccurthroughoutthesequence.
Despiteallofthenewandexcitingadvancesinfluorescentproteintechnology,mostoftheresearchgroupsaroundtheworldarestillusingthe“enhanced”versionofwild-typeGFP(termed EGFP),aswellastheoriginalcyanandyellowderivatives,asworkhorsesforamajorityofthepublishedinvestigationsincellbiology.Thereluctanceofmanyresearcherstotransitiontonewerfluorescentproteinvariantsisfueledbythecheckeredavailabilityoftheseproteins,coupledwith(thealltoooftenjustified)uncertaintiesaboutreportedclaimsofimprovedbrightness,photostability,monomericcharacter,andutilityinfusions.Inmanycases,justfindingasourceforanewfluorescentproteinorfusionvectorofpotentialinterestcanbeatime-consuminganddiscouragingchallenge.Thelackofdependablecommercialavailabilityoftenrequiresresearcherstorelyonthegenerosityoftheoriginalsourcelaboratory,whichinturn,canbebombardedwithanavalancheofrequestsshortlyafteranewprotein,biosensor,orfusionconstructhasbeenreported.Barringtheimplementationofanorganizedandefficientsystemforthedistributionoffluorescentproteinvariantstothescientificcommunity,thesituationisunlikelytoberemediedinthenearfuture.
ThoseresearcherswhoareultimatelyabletoobtainnewfluorescentproteinderivativescanbeseriouslydisappointedwiththeresultswhentheycomparethenewproteintotheirstandardGFPderivatives.Thefusionconstructsareoftenhamperedbyaggregation,lowbrightnesslevels,andrapidphotobleaching,ormaynottargetaparticularorganelleassuccessfullyastheclassical Aequorea GFPvariants.Itisnotunusualfortheproteindeveloperstogetfeedbackintheform:
“thisnew(chooseacolor)proteinformsaggregatesinthecytoplasm,doesn'tformthecorrect(chooseatarget)structureaswell,andismuchdimmerthanourotherGFPconstructs--wecan'treallyuseitformulticolorimagingasweexpected.Doyouknowofsomethingbetter?
”Actually,thesituationisnotuniversallythisdismalandthenewfluorescentproteinderivatives,whichseemtobereportedeveryseveralweeks,holdtremendouspromiseasversatilereportersforadvancedimagingtechnologiesinanumberofarenas,regardlessofthevariouspotentialpitfalls.
backtotop^GreenFluorescentProteinStructure
Amongthemostnoteworthyattributesoftheoriginalgreenfluorescentproteinderivedfromthe Aequoreavictoria jellyfish,aswellasthemorerecentlydevelopedpaletteofcolor-shiftedgeneticvariants,isthatastableandhighly-definedcylindricalpolypeptidestructureisessentialforthedevelopmentandmaintenanceoffluorescenceinthisveryremarkablefamilyofproteins.Theprinciplefluorophore(mostoftentermedachromophore)inGFPisatripeptideconsistingoftheresiduesserine,tyrosine,andglycineatpositions65-67inthesequence.Althoughthissimpleaminoacidmotifiscommonlyfoundthroughoutnature,itdoesnotgenerallyresultinfluorescence.Thechromophoreformsspontaneouslyaftertranslationwithouttherequirementforcofactorsorexternalenzymecomponents(otherthanmolecularoxygen),throughaself-catalyzedintramolecularrearrangementofthetripeptidesequencetoproducethefluorescentspecies(asillustratedinFigures1and3forthenaturallyoccurringwild-typeandenhancedGFPvariant,respectively).
PresentedinFigure3isaschematicdiagramofthechromophoreformationinmaturingenhancedgreenfluorescentprotein(EGFP),aderivativethatsubstitutestheaminoacidthreonineforserineatposition65(S65T).InFigure3(a),thepre-maturationEGFPfluorophoretripeptideaminoacidsequence(Thr65-Tyr66-Gly67)isstretchedintoalinearconfigurationsothatthethreonineresidueispositionedintheupperleft-handcornerofthediagram.Thefirststepinthematurationsequenceisthoughttobeaseriesoftorsionalpeptideandside-chainbondadjustmentsthatrelocatethecarboxylcarbonoftheserineaminoacidatposition65(Ser65)incloseproximitytotheaminonitrogenatomoftheglycineresidueatposition67(Gly67;seeFigures3(b)and3(c)).Nucleophilicattackbythiscarbonatomontheamidenitrogenofglycine,followedbydehydration,resultsintheformationofaconjugatedimidazolin-5-oneheterocyclicringsystem(Figure3(c)).Fluorescenceoccurswhenoxidationofthetyrosine(Tyr66)alpha-beta carbonbondbymolecularoxygenextendselectronconjugationoftheimidazolineringsystemtoincludethetyrosinephenylringanditspara-oxygensubstituent(Figure3(d)).Theresultisahighlyconjugatedπ-electronresonancesystemthatlargelyaccountsforthespectroscopicpropertiesoftheprotein.Extensivemutagenesisstudiessuggestthattheglycineresidueatposition67iscriticalinformationofthechromophore,andindeed,nofluorescentproteinshaveyetbeendiscoveredthatlackthisessentialelement.
Theprotective beta-barrel(describedinmoredetailbelow)structureprovidesascaffoldsurroundingthefluorophoretomaintainplanarityandfosterawiderangeofinteractionswithtrappedwatermoleculesandaminoacidsidechainsfromthepolypeptidebackbone.Combinedwiththeshortportio
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