In-Cell Double Electron − Electron Resonance at Nanomolar Protein Concentrations - Kucher et al. - 2021 - Unknown

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pubs.acs.org/JPCLLetterIn-CellDoubleElectron−ElectronResonanceatNanomolarProteinConcentrationsSvetlanaKucher,ChristinaElsner,MariyaSafonova,StefanoMaffini,andEnricaBordignon*CiteThis:J.Phys.Chem.Lett.2021,12,3679−3684ReadOnlineACCESSMetrics&MoreArticleRecommendations*sıSupportingInformationABSTRACT:Electronparamagneticresonance(EPR)spectroscopyisanestablishedtechniquetosite-specificallymonitorconformationalchangesofspin-labeledbiomolecules.Emergingin-cellEPRapproachesaimingtoaddressspin-labeledproteinsintheirnativeenvironmentstillstruggletoreachabroadapplicabilityandtotargetphysiologicallyrelevantproteinconcentrations.Here,wepresentacomparativeinvitroandin-celldoubleelectron−electronresonance(DEER)studydemonstratingthatnanomolarproteinconcentrationsareatreachtomeasuredistancesupto4.5nmbetweenproteinsitescarryingcommercialgadoliniumspinlabels.tudyingthestructureandfunctionofaproteininitsnativeMDa(StefanoMaffini,personalcommunication)todifferent15Senvironmentisessentialinmodernstructuralbiologytorecipientcells.Alternatively,proteinscanbedirectly16revealhowbiomoleculesreallyworkincells.Todate,afewsynthesizedwithgeneticallyencodedparamagneticmarkersprominentbiophysicaltechniquescanbeusedtogetinsights17−19ordirectlylabeledincells.Thelattertwomethodsareintheinnerworkingsofacell,namely,in-cellNMR,cellularratherchallengingandcannotyetbeappliedasaroutinecryo-electrontomography,andin-cellsingle-moleculeFörsterprocedure.Afewmethodsdesignedforin-cellEPRof1resonanceenergytransfer(FRET)spectroscopy.Additionally,membraneproteinsalsoexist,includingspinlabelingoftheelectronparamagneticresonance(EPR)spectroscopyisouterE.colicellmembraneproteins6,7,20ortheuseofspin-rapidlytransformingfromapurelyinvitrotechniquetowardlabelednanobodiestargetingaspecificmembraneprotein,theapromisingin-cellmethodology.Sincemostbiomoleculesarelatterbeingprovensofaronlyininside-outvesicles.5naturallydiamagnetic,site-directedspin-labeling(SDSL)isAnalysisofproteinsinsertedintolivingcellsentailsmultipleusuallyutilizedfortheattachmentofparamagneticmarkersatchallenges.First,thedeliveredproteinsshouldnotaggregate,DownloadedviaUNIVOFNEWMEXICOonMay16,2021at10:17:23(UTC).2thesitesofinterest.Pulseddipolarspectroscopy(PDS)iswhichisoftenthecasewhenhighmicromolarconcentrationsusedtomeasureinterspindistancesinbiomolecules,providingareused,andtheyshouldlocalizecorrectlytotheircellularSeehttps://pubs.acs.org/sharingguidelinesforoptionsonhowtolegitimatelysharepublishedarticles.insightsintotheirstructuralproperties.Doubleelectron−compartment.Anadditionalchallengeisthepresenceofelectronresonance(DEER,alsoknownasPELDOR)isoneofreducingagentsincells.Nitroxidespinlabels,whichremaintothecommonPDSmethodstodetermineinterspindistancedatethefirstchoiceforinvitroSDSLEPRstudies,arereduced3,4distributionsfrom1.5to∼10nm.DEERcanberoutinelyinthecell.17,21−23Chemicallyshieldednitroxidescanpartiallyappliedtospin-labeledproteinsdowntoafewmicromolarsustainthereducingcellenvironment,buttheirlifetimeisstillconcentrationsevenunderphysiologicalenvironments(see,limited.22,24Trityllabels,despitebeingbulkier,mayofferaforexample,refs5−7).validalternative.25−27Todate,spinlabelsbasedonForin-cellEPRapplications,afewstrategieshavebeenbiocompatiblechelatedGd3+complexeshavefoundthelargesttested.Themostcommonmethodimpliesinvitrolabelingofaapplicationforin-cellPDSstudiesofproteinsatmicromolarwater-soluble,recombinantlyproducedbiomoleculefollowedconcentrations.8,9,13,14,28−30byitsdeliverytothetargetcell.Severaldeliverymethodshavebeensuccessfullyappliedsuchaselectroporation,hypotonicswelling,andmicroinjectionsintooocytes.8−14ElectroporationReceived:January6,2021isawell-established,high-throughput,batchdeliverytechniqueAccepted:March23,2021thatdoesnotrequirespecifichandlingskillsorinstrumentalsetPublished:April8,2021up,anditissuitableforefficientandhomogeneousdeliveryofwater-solubleproteinsofdifferentshapesandsizesrangingfrompeptidestomultisubunitproteincomplexesupto1to2©2021TheAuthors.PublishedbyAmericanChemicalSocietyhttps://doi.org/10.1021/acs.jpclett.1c000483679J.Phys.Chem.Lett.2021,12,3679−3684

1TheJournalofPhysicalChemistryLetterspubs.acs.org/JPCLLetterFigure1.PrimaryDEERtracesV(t)/V(0)anddistancedistributionsP(r)obtainedbyTikhonovanalysis(cyan)andDEERNet(black,withshadedgrayareasrepresentingtheuncertainty)forBid(A)andT4lysozyme(B)labeledwithMTSL(leftpanels,50K)andGd-DOTA-maleimide(rightpanels,10K).Protein(andspin)concentrations,signal-to-noiseratio(SNR),andaccumulationtimeareindicated.AsterisksdenoteadistanceartifactcreatedbyTikhonovanalysis.TheoverallproteinamountinthecytosolofmammalianSeveralinvitronanomolarPDSstudieshavebeenrecently31cellsisintheorderof200−300mg/mL;however,differentdemonstrated.Bodeandco-workershaveshownthe35proteinsexistatconcentrationswhichcanvaryfromhighapplicabilityof100nMCu−CuRIDMEand100nM36micromolar(e.g.,α-synucleininneuronalsynapses:5−50nitroxide−nitroxidePELDORusingacommercialQ-band32,33μM)tolownanomolar(e.g.,pro-apoptoticendogenousspectrometeronbiologicalsystemsinvitro.Schiemannandco-34Bax:3−170nMinhumancells).Detectinginterspinworkershavesynthesizednewtrityllabelsenablingdetectiondistancesatnanomolarconcentrationsincellsischallengingofdistancesat45−90nMconcentrationsinvitroand11μM25,26forPDSmethodsbutwouldpavethewayforabroaderin-cellviaQ-bandDQCmeasurements.However,uptoapplicabilityofthemethodology.Infact,insertingproteinsinnow,submicromolarin-cellconcentrationshavenotyetbeencellsathigherthanphysiologicalconcentrationscanaltertheachievedwithanyPDStechniqueinconjunctionwithanyfractionofexistingproteincomplexeswhichhavenano-to-label.micromolardissociationconstantsandcanleadtoproteinThepresenceofnaturallyoccurringparamagneticspeciesmislocalization,uncontrolledaggregation,formationofin-insidethecell,andinparticularofMn2+ions,isanotherclusionbodies,andcelldeath.limitingfactorforthesensitivityofin-cellPDS,asshownfor3680https://doi.org/10.1021/acs.jpclett.1c00048J.Phys.Chem.Lett.2021,12,3679−3684

2TheJournalofPhysicalChemistryLetterspubs.acs.org/JPCLLetterFigure2.High-resolutionimagesofHEKcellsafterelectroporationofAlexa647-labeledT4lysozymetakenafter6h(A)andovernight(B)recoveryat37°C.Asaconsequenceoftheshortrecoverytime,someofthecellsin(A)havenotcompletedtheadherenceprocessandthereforedonotdisplaythetypicalelongatedmorphologyofadherentcells.However,asanindicationofgoodhealth,cellsinoursampledisplayhigh15viabilityrates(>80%,seeTableS6),whichisinagreementwithpreviousreports.3+28,29Gdlabels.Itisdifficulttocontrolthemanganesecontenttime.Mostimportantly,despitethelowerSNR,themainpeaksincellsbecauseitisnotdirectlysupplementedinthecellextractedatnanomolarconcentrationsremainedreliable,andgrowthmedia.BothGd3+andMn2+arehighspintransition-acceptableSNRsintherange8−22werereachedin13−48h.metalions(spinof7/2and5/2,respectively),exhibitingNotably,Gdlabelsalloweddetectionoflongertimetraceswithsimilarrelaxationpropertiesandspectraloverlap.Therefore,respecttoMTSLduetofavorablerelaxationtimes(seeFiguresufficientMnsignalsuppressionisnotpossible.S3)andhigherechosignalintensityatcomparableHere,weexplorethepossibilitiesandlimitationsofaconcentrations.Oneadvantageofusingsampleswithlow-to-commercialstate-of-the-artQ-bandDEERsetuptoevaluatesubmicromolarspinconcentrationsisthatthebackgroundinterspindistancesinbiomoleculesatnanomolarconcen-functionoftheDEERtracebecomesalmostflat(Figure1),trations.WeshowthatDEERcanbeappliedtoproteinsintherebyfacilitatingdataanalysis.Intriguingly,theintermolec-cellsatnanomolarconcentrationsontheexampleofT4ularbackgrounddecaywasfoundtobemorepronouncedthanlysozymeelectroporatedintohumanembryotickidney(HEK)expectedforthe300nMGd-labeled-Bid(Figure1A),whichcells.Tothisend,weusedastandardelectroporationprotocolmayindicatepartialproteinaggregationinthesample.with∼10timesfewerstartingmaterialascomparedtotheTotesttheelectroporationmethodatthelowestachievable9,14,28,29literature,pavingthewaytowardin-cellDEERforaproteinconcentrations,wechosegadolinium-labeledT4broaderrangeofbiomolecularsystems.lysozymeasamodelsystem,sinceBidisapro-apoptoticInthefollowing,wefirstaddressthereliabilityandfeasibilityproteinandmightinduceapoptosisincells,whichisofDEERatdecreasingproteinconcentrationsinvitro.Weuseddetrimentalfortheintendedstudy.First,differentcelllines24bacteriophageT4lysozymelabeledatpositions72and131weretestedtoidentifythemostsuitableones(FigureS7).37,38andthepro-apoptoticBcl-2proteinBidlabeledatthetwoHeLacells,derivedfromcervicalcancercells,providehighnativecysteines(positions30and126)withMTSLorGd3+-viabilityanddeliveryratesinelectroporationexperimentsand9,14,28,29DOTA-maleimide(seeSISection1fordetails).Thesampleshavepreviouslybeenusedforin-cellEPR.Here,wewerepreparedincommonlyusednondeuteratedaqueouscomparedthemwithhumanembryotickidneyHEK293cells,buffers,andpriortofreezing,deuteratedglycerol(30−50%v/humanboneosteosarcomacellsU2OS,andretinalpigmentv)wasaddedasacryoprotectanttoprolongthephasememoryepithelialcelllineRPE1,allsuitableforelectroporation15time.WechosetheGd-DOTA-maleimidecomplexasitisexperiments.Interestingly,allfourtestedmammaliancellbiocompatible,commerciallyavailable,suitableforcysteine-linesrevealedMn-dominatedecho-detectedfieldsweptbasedlabeling,andthereforehasabroadapplicability.Figure1(EDFS)spectrawithcomparableintensitypercell(FigureshowstheDEERtracesobtainedonspin-labeledBidatS7,TableS3).However,wefoundthatHEKcellsaredecreasingconcentrationsfrom30μMto300nMandonspin-advantageousfortheelectroporationexperimentsbecauseoflabeledT4lysozymefrom12μMto120nM.Thechoseneasygrowthandlessadherentarchitectureascomparedtomaximalproteinconcentrationproducesinashorttimeanothertestedlines,whichallowsforeasierhandlingandshorter3841,42excellentsignal-to-noiseratio(SNR),definedastherecoverytime.modulationdepthoftheDEERtrace(Δ)dividedbytheWeusedanelectroporationprotocolwhichwaspreviously9,14,29standarddeviationofthenoise(seeFigure1A).ThelowestusedforEPRbutdecreasedthestartingproteinproteinconcentrations(∼120−300nM)approachtheconcentrationsbyoneorderofmagnitude(protocolgiveninphysiologicalconcentrationsofpro-apoptoticproteinsinSISection4).WedeliveredAlexa647-labeledT4lysozymeat34cells.SimilarSNRscouldbeobtainedforbothlabels(FigureCstart=5.6μMorGd-T4lysozymeatCstart=11.0μMinto1);however,fortheGd3+spinlabelsthemodulationdepthHEKcells.TheAlexa-labeledproteinswereusedtoassessthe(Δ)issmallerandthewidthofthedistancedistributionisinternalizationandlocalizationoftheproteinandtheGd-larger.labeledproteinsforin-cellDEER(Figure2).ToanalyzetheDEERtracesweusedtheTikhonovMicroscopyimagestakenafter6hrecoverytimeshoweda39regularizationmethodandaneuralnetworkanalysishomogeneousproteinlocalizationwithinthecytosoland39,40(DEERNet)availableinDeerAnalysis2019(Figure1,nucleuswithonlyminorfactionsofaggregates,recognizableasFigureS6).Theobtaineddistancedistributionswereinbrightspots(seeFigure2A).Inagreementwithprevious15excellentagreement,exceptforthelong-distanceartifactreports,thecellsshowedahighcellviability(>80%,see(denotedwithasterisksinFigure1),whichappearedinTableS6).However,afterovernightrecovery,thefluorescentTikhonovanalysisofDEERtraceswithshortdipolarevolutionintensitydecreasedandonlyaggregateswerefound(Figure3681https://doi.org/10.1021/acs.jpclett.1c00048J.Phys.Chem.Lett.2021,12,3679−3684

3TheJournalofPhysicalChemistryLetterspubs.acs.org/JPCLLetterFigure3.Q-bandecho-detectedfieldswept(EDFS)spectraof(A)0.4μMGd-DOTA-maleimideinvitroandtwomockelectroporationHEKsamplesresuspendedindeuteratedornondeuteratedbuffer,and(B)anHEKcellpelletcontainingGd-labeledT4lysozymeanditssupernatant.TheB-fieldaxisisfrequency-normalizedforbetterspectralcomparison.Spectradetectedat10K.2B),possiblyduetothedegradationofT4lysozymeinhuman43cellswith2−10hhalf-life.Thus,tominimizeproteins’degradation,weoptedfor3hrecoverytime.Figure3Apresentstheecho-detectedfieldswept(EDFS)spectrumofHEKcellselectroporatedwithbufferalone(mocksample),characterizedbythetypicalsixhyperfinelinesofMn2+(blueandcyan).Thecellspectraarecomparedtothespectrumofa0.4μMGd-DOTA-maleimidestocksolutiontohighlighttherelativepositionofthemaximumofthegadoliniumspectrum.Figure3B(violet)showstheEDFSspectrumdetectedontheHEKcellselectroporatedwithGd-Figure4.Nanomolarin-cellDEERonGd-T4lysozymeelectro-T4lysozyme(seeSISection6fordetails).poratedintoHEKcells.(A)DEERprimarydatadetectedat10KandAsacontrol,wedetectedtheEDFSspectrumofthe(B)distancedistributionwithuncertainties(shadedarea)obtainedbysupernatantcollectedafterthelastwashingstep,whichrevealsDEERNetinDeerAnalysis2019.ThedataobtainedonGd-T4onlytheEPRcavitybackgroundsignal(grayinFigure3B).lysozymesampleselectroporatedintoHEKcells(violet,2.9μsThisconfirmedthattheGd3+signaldetectedintheHEKcellsdipolarevolutiontime,SNR19in48h)arecomparedtothedataarisessolelyfrominternalizedproteins.Tocalculatetheobtainedinvitroon12μMproteinsolution(black,fromFigure1B).concentrationoftheGd3+spinsintheelectroporatedHEKcells,wereconstructedthespectrumincells(violetinFigureThemodulationdepthandthemeandistancewerefully3B)addingtheweightedspectraofthemocksample(blueinreproducedinasecondbiologicalrepeat(FigureS10).ThisFigure3A)andoftheGd-DOTA-maleimidesolution(blackinrepeatcontainednondeuteratedbufferandcryoprotectant,Figure3A).provingthatitisalsopossibletoachievein-cellnanomolarFromthespectralreconstruction(dottedlineinFigure3B)sensitivitywithoutdeuterationofthebuffer,attheexpenseofweobtaineda182±33nMbulkgadoliniumspinthesignal-to-noiseratio.concentrationintheDEERtube,whichcorrespondsto101Thesmallshoulderinthedistancedistributionataround3±21nMconcentrationofT4lysozyme.Knowingthetotalnmischaracterizedbylargeuncertaintiesandwasnotnumberofcellsandthevolumeinthetube(1.2×107cellsinreproducible;therefore,itcannotbeunambiguouslyinter-40μL,fordetailsseetheSI)andassuminganaveragevolumepreted(FigureS10).Notably,weconfirmedtheintegrityoftheof(990±165)femtoliters(i.e.,10−15liters)fortheHEKcellsmeasuredbyDEER(Figures4andS10)bycellviability44,45cells(seeSISection7),thiswouldcorrespondtoananalysisperfomedafterthawingthesample(SISection7,averageconcentrationofabout340±92nMincell.TableS6).Thein-cellDEERtracerecordedontheelectroporatedTherelativelysmallzero-fieldsplitting(ZFS)ofgadoliniumsampleisshowninFigure4A(violet).TheminimaldecayofchelatedbyDOTAisoneofthepropertiesthat,inthebackgroundfunctionconfirmstheabsenceofproteincombinationwithcapabilitiesofastate-of-the-artQ-bandaggregationinHEKcells.Inlinewithpreviouslyreportedspectrometerandaresonatorallowinglargesamplevolumes14,292+data,duetothepresenceofthebackgroundMnsignal,(40μL),enabledustoachievesuchunprecedentsensitivityinthemodulationdepthofthein-cellDEERtracewaslowerthancells.However,thedisadvantageofthisGd-basedlabelisathatinvitro(1.7%vs5.6%).Thedistancedistributionobtainedhighflexibilityofthelinker,leadingtointerspindistancesincells(Figure4B)atnanomolarproteinconcentrationbroaderthanthoseobtainedwiththeconventionalMTSLcorrelateswelltothatobtainedinvitro,withameaninterspinlabels.Therefore,alowerdistanceresolutionisexpectedasdistancebetweenthetwolabeledsitesof4.5nm.Thiscomparedtonitroxides(seeFigure1).ThesynthesizedrigidindicatesthatT4lysozymeadoptsthesameglobularlinkersforGdchelatorsreportedinrefs14and28have∼3conformationinvitroandin-cell.timeslargerwidthofthecentraltransitionascomparedtothe3682https://doi.org/10.1021/acs.jpclett.1c00048J.Phys.Chem.Lett.2021,12,3679−3684

4TheJournalofPhysicalChemistryLetterspubs.acs.org/JPCLLettercommercialDOTA-maleimide(180MHzvs50MHz).Thus,■ACKNOWLEDGMENTSweexpectthat,withthesetuppresentedhere,thelowestTheauthorswouldliketoacknowledgeStefanRaunserandachievableconcentrationwillbeabout3timeshigherthanthatDanielRoderer(MPIofMolecularPhysiology,Dortmund)forobtainedherewithcommercialGd-DOTAmaleimidelabels.fruitfuldiscussionandinitialexperimentsandBeateVoßandInconclusion,webenchmarknanomolarinvitroandin-cellJanineBeermannfortechnicalassistance.E.B.wouldliketoDEERwithGd-labeledproteins,describinganelectroporationthankAndreaMusacchio(MPIforMolecularPhysiology,protocolutilizingHEKcells,whichrequires10timeslessspin-Dortmund)forhissupporttostarttheprojectandGunnarlabeledproteincomparedtoavailablestate-of-the-artproto-Jeschke(ETHZurich)forthehomemadeQ-bandresonator.cols.LoweringtheintracellularconcentrationofthedeliveredThisworkisfundedbytheDeutscheForschungsgemeinschaftproteinlimitsproteinaggregationand,mostimportantly,paves(DFG,GermanResearchFoundation)underGermany′sthewayforthestructuralanalysisofproteinsattheirExcellenceStrategy(EXC2033-390677874)RESOLVandphysiologicalconcentrationsinnativeenvironment.WeshowbytheEuropeanResearchCouncil(669686)(S.M.).thatfourdifferentmammaliancelllinesexhibitcomparableintracellularmanganeseconcentrations;therefore,theachieved■sensitivityisalsoexpectedforothermammaliancells.REFERENCESHowever,wefoundthatHEKcellsareparticularlysuitedfor(1)Ito,Y.;Selenko,P.Cellularstructuralbiology.Curr.Opin.Struct.Biol.2010,20(5),640−648.electroporationexperimentbecausetheirweakadhesiontothe41,42(2)Hubbell,W.L.;Lopez,C.J.;Altenbach,C.;Yang,Z.cultureplasticdishfacilitateshandlingofthesampleandTechnologicaladvancesinsite-directedspinlabelingofproteins.analysisaftershorterrecoverytimes.Notably,suspensionHEKCurr.Opin.Struct.Biol.2013,23(5),725−733.46,47celllinederivativesarealsoavailable,e.g.,HEK293-F,(3)Pannier,M.;Veit,S.;Godt,A.;Jeschke,G.;Spiess,H.W.Dead-whichcouldofferfurtheradvantagesforin-cellEPR.Wetimefreemeasurementofdipole-dipoleinteractionsbetweenelectronbelievethatthepresentedelectroporationmethodologyforspins.J.Magn.Reson.2000,142(2),331−340.Gd-labeledwater-solubleproteinswillbroadentheapplication(4)Jeschke,G.DEERdistancemeasurementsonproteins.Annu.Rev.Phys.Chem.2012,63,419−446.ofin-cellDEERandwillenableresearcherstoobtainnew(5)Galazzo,L.;Meier,G.;Timachi,M.H.;Hutter,C.A.J.;Seeger,insightsintheconformationalplasticityofproteinsintheirM.A.;Bordignon,E.Spin-labelednanobodiesasproteinconforma-nativemilieuatphysiologicallyrelevantconcentrations.tionalreportersforelectronparamagneticresonanceincellularmembranes.Proc.Natl.Acad.Sci.U.S.A.2020,117(5),2441−2448.■(6)Nyenhuis,D.A.;Nilaweera,T.D.;Niblo,J.K.;Nguyen,N.Q.;ASSOCIATEDCONTENTDuBay,K.H.;Cafiso,D.S.EvidencefortheSupramolecular*sıSupportingInformationOrganizationofaBacterialOuter-MembraneProteinfromInVivoTheSupportingInformationisavailablefreeofchargeatPulseElectronParamagneticResonanceSpectroscopy.J.Am.Chem.https://pubs.acs.org/doi/10.1021/acs.jpclett.1c00048.Soc.2020,142(24),10715−10722.(7)Ketter,S.;Gopinath,A.;Rogozhnikova,O.;Trukhin,D.;ExperimentaldetailsandadditionalsupportingfiguresTormyshev,V.M.;Bagryanskaya,E.G.;Joseph,B.InSituLabelingandtables(PDF)andDistanceMeasurementsofMembraneProteinsinE.coliUsingFinlandandOX063TritylLabels.Chem.-Eur.J.2021,27,2299−2304.■(8)Martorana,A.;Bellapadrona,G.;Feintuch,A.;DiGregorio,E.;AUTHORINFORMATIONAime,S.;Goldfarb,D.ProbingproteinconformationincellsbyEPRCorrespondingAuthordistancemeasurementsusingGd3+spinlabeling.J.Am.Chem.Soc.EnricaBordignon−RuhrUniversityBochum,Facultyof2014,136(38),13458−13465.ChemistryandBiochemistry,44801Bochum,Germany;(9)Theillet,F.X.;Binolfi,A.;Bekei,B.;Martorana,A.;Rose,H.M.;orcid.org/0000-0003-2450-5161;Stuiver,M.;Verzini,S.;Lorenz,D.;vanRossum,M.;Goldfarb,D.;Selenko,P.Structuraldisorderofmonomericalpha-synucleinpersistsEmail:enrica.bordignon@rub.deinmammaliancells.Nature2016,530(7588),45−50.(10)Krstic,I.;Hansel,R.;Romainczyk,O.;Engels,J.W.;Dotsch,V.;AuthorsPrisner,T.F.Long-rangedistancemeasurementsonnucleicacidsinSvetlanaKucher−RuhrUniversityBochum,FacultyofcellsbypulsedEPRspectroscopy.Angew.Chem.,Int.Ed.2011,50ChemistryandBiochemistry,44801Bochum,Germany(22),5070−5074.ChristinaElsner−RuhrUniversityBochum,Facultyof(11)Azarkh,M.;Singh,V.;Okle,O.;Seemann,I.T.;Dietrich,D.R.;ChemistryandBiochemistry,44801Bochum,GermanyHartig,J.S.;Drescher,M.Site-directedspin-labelingofnucleotidesMariyaSafonova−RuhrUniversityBochum,Facultyofandtheuseofin-cellEPRtodeterminelong-rangedistancesinaChemistryandBiochemistry,44801Bochum,Germanybiologicallyrelevantenvironment.Nat.Protoc.2013,8(1),131−147.StefanoMaffini−MaxPlanckInstituteofMolecular(12)Dunkel,S.;Pulagam,L.P.;Steinhoff,H.J.;Klare,J.P.InvivoPhysiology,DepartmentofMechanisticCellBiology,44227EPRonspinlabeledcolicinArevealsanoligomericassemblyofthepore-formingdomaininE.colimembranes.Phys.Chem.Chem.Phys.Dortmund,Germany2015,17(7),4875−4878.Completecontactinformationisavailableat:(13)Qi,M.;Groß,A.;Jeschke,G.;Godt,A.;Drescher,M.Gd(III)-https://pubs.acs.org/10.1021/acs.jpclett.1c00048PyMTALabelIsSuitableforIn-CellEPR.J.Am.Chem.Soc.2014,136(43),15366−15378.Notes(14)Yang,Y.;Yang,F.;Gong,Y.-J.;Chen,J.-L.;Goldfarb,D.;Su,X.-C.AReactive,RigidGdIIILabelingTagforIn-CellEPRDistanceTheauthorsdeclarenocompetingfinancialinterest.MeasurementsinProteins.Angew.Chem.,Int.Ed.2017,56(11),Allprimarydataofthefiguresinthemaintextcanfoundat2914−2918.https://git.noc.ruhr-uni-bochum.de/bordie2h/nanomolar-in-(15)Alex,A.;Piano,V.;Polley,S.;Stuiver,M.;Voss,S.;Ciossani,cell-deer.G.;Overlack,K.;Voss,B.;Wohlgemuth,S.;Petrovic,A.;Wu,Y.;3683https://doi.org/10.1021/acs.jpclett.1c00048J.Phys.Chem.Lett.2021,12,3679−3684

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