Table Of Content

Methods in Molecular Biology 2564 Mayank Sharma Editor Fluorescent Proteins Methods and Protocols M M B ETHODS IN OLECULAR IO LO GY SeriesEditor JohnM.Walker School of Lifeand MedicalSciences, University ofHertfordshire Hatfield, Hertfordshire, UK Forfurther volumes: http://www.springer.com/series/7651 For over 35 years, biological scientists have come to rely on the research protocols and methodologiesinthecriticallyacclaimedMethodsinMolecularBiologyseries.Theserieswas thefirsttointroducethestep-by-stepprotocolsapproachthathasbecomethestandardinall biomedicalprotocolpublishing.Eachprotocolisprovidedinreadily-reproduciblestep-by- step fashion, opening with an introductory overview, a list of the materials and reagents neededtocompletetheexperiment,andfollowedbyadetailedprocedurethatissupported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitutethekeyingredientineachandeveryvolumeoftheMethodsinMolecularBiology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexedinPubMed. Fluorescent Proteins Methods and Protocols Edited by Mayank Sharma Institute of Molecular Plant Biology, ETH Zürich, Zürich, Switzerland Editor MayankSharma InstituteofMolecularPlantBiology ETHZu¨rich Zu¨rich,Switzerland ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-0716-2666-5 ISBN978-1-0716-2667-2 (eBook) https://doi.org/10.1007/978-1-0716-2667-2 ©TheEditor(s)(ifapplicable)andTheAuthor(s),underexclusivelicensetoSpringerScience+BusinessMedia,LLC,part ofSpringerNature2023 Thisworkissubjecttocopyright.AllrightsaresolelyandexclusivelylicensedbythePublisher,whetherthewholeorpart of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting,reproductionon microfilmsorinanyotherphysicalway,andtransmissionorinformation storageand retrieval,electronicadaptation, computersoftware,orbysimilar ordissimilar methodologynow knownorhereafter developed. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnotimply, evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsandregulations andthereforefreeforgeneraluse. Thepublisher,theauthors,andtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedto betrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty, expressedorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeen made.Thepublisherremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations. ThisHumanaimprintispublishedbytheregisteredcompanySpringerScience+BusinessMedia,LLC,partofSpringer Nature. Theregisteredcompanyaddressis:1NewYorkPlaza,NewYork,NY10004,U.S.A. Preface Thediscoveryoffluorescentprotein(FP)hasrevolutionizedthewaywestudycellbiology.A largerepertoireoffluorescentproteinshasbeendevelopedinpastyearsandconcurrentlythe FP-basedexperimentalapproachestostudythebehaviorandfunctionofproteinswithinand outsidethecellularsystems.ThisvolumeoftheMethodsinMolecularBiologyseriesbrings together some cutting-edge laboratory protocols to characterize the novel fluorescent proteins and approaches based on fluorescent proteins to answer some of the key cell biologicalquestions. Tobeginwith,thefirstchapterinthisvolumedescribesarecentlydevelopedfluorescent proteindatabasecalledFPbaseandprovidesacomprehensiveguidethatenablestheusersto maximize its potential. The second and third chapters explain the essentials of selecting fluorescentproteinsforimagingexperimentsandadvancedapproachestostudyfluorescent protein dynamics and behavior in vivo. The fourth and fifth chapters elaborate on the protocol for developing new fluorescent proteins via directed evolution and a method to determine their molecular structures, respectively. The next two chapters in this volume provide an all-inclusive guide to designing and studying novel Flavin-based fluorescent proteins (FbFPs), holding a strong potential for studying biological systems where oxygen availabilityislimited. SubsequentchapterscontainmethodsthatshowcasetheadaptationofFPstoadiverse range of biological systems. Chapter 8 provides an experimental guide for endogenous proteinlabelingin humancell lineswith therecentlydeveloped CRISPR/Cas9-basedself- complementing split-GFP system. Chapter 9 details protocols to express fluorescent proteins in plant cells for determining the subcellular protein localization. The next chapter describes an approach for expressing fluorescent protein fusions in yeast cells and to quantitativelyassesstheirimpactoncellgrowth.Thefollowingchapterisbasedonarecently developed method to synthesize DNA-binding fluorescent proteins and a technique to visualizeDNAatthesingle-moleculelevel. In parallel, fluorescent proteins have been extensively modified as sensor molecules for detectingand/orquantifyingotherbiomolecules.MostoftheseFP-basedbiosensorswork on the principles of either allosteric modulation of protein characteristics or fo¨rster reso- nanceenergytransfer(FRET).As follows,Chap.12ofthisvolume describesaprotocol to determine intracellular citrate concentration with the citrate responsive biosensors, namely Citron1 and Citroff1. Another chapter describes a redox-sensitive fluorescent protein, roGFP, and a method to determine its redox status with a gel-based approach. Likewise, Chap. 14 details the method to determine oxygen dynamics in yeast cells with luciferase- based biosensors and discusses the possible approach to adapt this system to fluorescent proteins. v vi Preface Thelastthreechaptersofthisvolumeexhibithowfluorescentproteinsareutilizedina diverse range of applications in cell biology, for example, in correlative light and electron microscopy (CLEM) imaging, in determining the thermostability of membrane proteins, and determination of protein-protein interactions. Overall, this book provides a wealth of practicalresourcesforscholarsinterestedinusingfluorescentproteinsintheir research. Zu¨rich,Switzerland MayankSharma Contents Preface ..................................................................... v Contributors................................................................. ix 1 UsingFPbase:TheFluorescentProteinDatabase.... ........ ....... ........ 1 TalleyJ.Lambert 2 SelectingtheFluorescentProteinforInVivoImagingExperiments........... 47 MayankSharma 3 AdvancedFluorescenceMicroscopyMethodstoStudyDynamics ofFluorescentProteinsInVivo...... ....... ....... ........ ....... ........ 53 MarcelaDıázandLeonelMalacrida 4 DirectedEvolutionofFluorescentProteinsinBacteria ....... ....... ........ 75 SaraMattson,GeraldineN.Tran,andErikA.Rodriguez 5 FluorescentProteins:Crystallization,StructuralDetermination, andNonnaturalAminoAcidIncorporation ....... .. ...... .. ...... ......... 99 RochelleD.Ahmed,HusamSabahAuhim,HarleyL.Worthy, andD.DafyddJones 6 DevelopmentandCharacterizationofFlavin-BindingFluorescentProteins, PartI:BasicCharacterization........ ....... ....... ........ ....... ........ 121 AlinaRemeeva,AnnaYudenko,VeraV.Nazarenko,OlegSemenov, AnastasiaSmolentseva,AndreyBogorodskiy,IvanMaslov,Valentin Borshchevskiy,andIvanGushchin 7 DevelopmentandCharacterizationofFlavin-BindingFluorescentProteins, PartII:AdvancedCharacterization.......... ....... ........ ....... ........ 143 NoraLisaBitzenhofer,FabienneHilgers,GabrielaN.Bosio, JoaquimTorra,GiorgiaCasini,FelixR.M.Beinlich, EstherKnieps-Gru¨nhagen,ValentinGordeliy,Karl-ErichJaeger, SantiNonell,UlrichKrauss,ThomasGensch,andThomasDrepper 8 CRISPR-Cas9-MediatedKnock-InApproachtoInserttheGFP Tag 11 intotheGenomeofaHumanCellLine ..... ...... .... ..... ....... ........ 185 RyoTamuraandDaichiKamiyama 9 FluorescentProtein-BasedApproachesforSubcellularProteinLocalization inPlants ........... ....... ........ ....... ....... ........ ....... ........ 203 MayankSharma,RalfBerndKlösgen,andBationaBennewitz 10 GrowthRateEvaluationoftheBuddingYeastSaccharomycescerevisiaeCells CarryingEndogenouslyExpressedFluorescentProteinFusions ...... ........ 213 KaraL.Schneider,LucasE.Reibenspies,ThomasNyström, andSviatlanaShashkova 11 DNAVisualizationUsingFluorescentProteins...... .... .. .. ....... ........ 223 XuelinJin,Y.TeheeKim,andKyubongJo vii viii Contents 12 QuantificationofIntracellularCitrateConcentrationswithGenetically EncodedBiosensors........ ........ ....... ....... ........ ....... ........ 247 YufengZhao,KeiTakahashi-Yamashiro,YiShen, andRobertE.Campbell 13 QuantificationofRedox-SensitiveGFPCysteineRedoxStateviaGel-Based Read-Out.......... ....... ........ ....... ....... ........ ....... ........ 259 FinjaBohle,AndreasJ.Meyer,andStefanieJ.Mueller-Schuessele 14 AssessingInVivoOxygenDynamicsUsingPlantN-TerminalDegrons inSaccharomycescerevisiae... ...... .. .. ..... ....... ........ ....... ........ 269 MikelLavilla-PuertaandBeatriceGiuntoli 15 Osmium-ResistantFluorescentProteinsandIn-ResinCorrelative Light-ElectronMicroscopyofEpon-EmbeddedMammalian CulturedCells...... ....... ........ ....... ....... ........ ....... ........ 287 IseiTanida,JunjiYamaguchi,Soichiro Kakuta, andYasuoUchiyama 16 Fluorescence-DetectionSize-ExclusionChromatography-Based ThermostabilityAssayforMembraneProteins....... ........ ... .... ........ 299 HebangYao,HongminCai,andDianfanLi 17 DeterminingProtein-ProteinInteractionwithGFP-TrapBeads ...... ........ 317 IremYilmazer,MelanieR.Abt,YuanyuanLiang,DavidSeung, SamuelC.Zeeman,andMayankSharma Index ...................................................................... 325 Contributors MELANIER.ABT • InstituteofMolecularPlantBiology,ETHZu¨rich,Zu¨rich,Switzerland ROCHELLED.AHMED • SchoolofBiosciences,MolecularBiosciencesDivision,Cardiff University,Cardiff,UK HUSAMSABAHAUHIM • DepartmentofBiology,CollegeofScience,UniversityofBaghdad, Baghdad,Iraq FELIXR.M.BEINLICH • InstituteofBiologicalInformationProcessingIBI-1:Molecularand CellularPhysiology,ForschungszentrumJu¨lichGmbH,Ju¨lich,Germany BATIONABENNEWITZ • InstituteofBiology-PlantPhysiology,MartinLutherUniversityHalle- Wittenberg,Halle(Saale),Germany NORALISABITZENHOFER • InstituteofMolecularEnzymeTechnology,HeinrichHeine UniversityDu¨sseldorf,ForschungszentrumJu¨lich,Ju¨lich,Germany ANDREYBOGORODSKIY • ResearchCenter forMolecularMechanismsofAgingand Age-RelatedDiseases,MoscowInstituteofPhysicsandTechnology,Dolgoprudny,Russia FINJABOHLE • InstituteofCropScienceandResourceConservation(INRES),Rheinische Friedrich-Wilhelms-Universita€tBonn,Bonn,Germany;MolecularBotany,Departmentof Biology,TUKaiserslautern, Kaiserslautern,Germany VALENTIN BORSHCHEVSKIY • ResearchCenter forMolecularMechanismsofAgingand Age-RelatedDiseases,MoscowInstituteofPhysicsandTechnology,Dolgoprudny,Russia GABRIELAN.BOSIO • InstituteofBiologicalInformationProcessingIBI-1:Molecularand CellularPhysiology,ForschungszentrumJu¨lichGmbH,Ju¨lich,Germany ROBERTE.CAMPBELL • DepartmentofChemistry,UniversityofAlberta,Edmonton,AB, Canada;DepartmentofChemistry,TheUniversityofTokyo,Tokyo,Japan GIORGIA CASINI • InstituteofBiologicalInformationProcessingIBI-1:Molecularand CellularPhysiology,ForschungszentrumJu¨lichGmbH,Ju¨lich,Germany MARCELADIÁZ • AdvancedBioimagingUnit,InstitutPasteurofMontevideo& UniversidaddelaRepu´blica,Montevideo,Uruguay THOMASDREPPER • InstituteofMolecularEnzymeTechnology,HeinrichHeineUniversity Du¨sseldorf,ForschungszentrumJu¨lich,Ju¨lich,Germany THOMASGENSCH • InstituteofBiologicalInformationProcessingIBI-1:Molecularand CellularPhysiology,ForschungszentrumJu¨lichGmbH,Ju¨lich,Germany BEATRICEGIUNTOLI • BiologyDepartment,UniversityofPisa,Pisa,Italy;Sant’AnnaSchool ofAdvancedStudies,Plantlab,Pisa,Italy VALENTIN GORDELIY • InstituteofBiologicalInformationProcessingIBI-7:Structural Biochemistry,ForschungszentrumJu¨lich,Ju¨lich,Germany;InstitutdeBiologieStructurale J.-P.Ebel,Universite´GrenobleAlpes-CEA-CNRS,Grenoble,France IVANGUSHCHIN • ResearchCenter forMolecularMechanismsofAgingandAge-Related Diseases,MoscowInstituteofPhysicsandTechnology,Dolgoprudny,Russia FABIENNEHILGERS • InstituteofMolecularEnzymeTechnology,HeinrichHeineUniversity Du¨sseldorf,ForschungszentrumJu¨lich,Ju¨lich,Germany KARL-ERICHJAEGER • InstituteofMolecularEnzymeTechnology,HeinrichHeineUniversity Du¨sseldorf,ForschungszentrumJu¨lich,Ju¨lich,Germany;InstituteofBio-andGeosciences IBG-1:Biotechnology,ForschungszentrumJu¨lichGmbH,Ju¨lich,Germany XUELINJIN • CollegeofAgriculture,YanbianUniversity,Jilin,China ix