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1、PROTOCOLSite-specificincorporationoffluorotyrosinesintotheR2subunitofE.coliribonucleotidereductasebyexpressedproteinligationMohammadRSeyedsayamdost1,CyrilSYee1&JoAnneStubbe1,21DepartmentofChemistry,2DepartmentofBiology,MassachusettsInstituteofTechnology,77MassachusettsAvenue,Cam
2、bridge,Massachusetts02139-4307,USA.CorrespondenceshouldbeaddressedtoJ.S.(stubbe@mit.edu).Publishedonline10May2007;doi:10.1038/nprot.2007.159sExpressedproteinligation(EPL)allowssemisynthesisofatargetproteinwithsite-specificincorporationofprobesorunnaturalaminoacidsatitsNorCtermi
3、ni.Here,wedescribetheprotocolthatourlabhasdevelopedforincorporatingfluorotyrosines(FnYs)atresidue356ofthesmallsubunitofEscherichiacoliribonucleotidereductaseusingEPL.Inthisprocedure,themajorityoftheprotein(residues1–353outof375)isfusedtoaninteindomainandpreparedbyrecombinantexp
4、ression,yieldingtheproteininanatureprotocol/thioester-activated,truncatedform.Theremainderoftheprotein,a22-merpeptide,ispreparedbysolid-phasepeptidesynthesisandmoc.containstheFnYatthedesiredposition.Ligationofthe22-merpeptidetothethioester-activatedR2andsubsequentpurificationer
5、yieldfull-lengthR2withtheFnYatresidue356.Theproceduretogenerate100mgquantitiesofY356FnY-R2takes3–4months.utan.wwINTRODUCTIONw//:Problemunderinvestigationref.14).FnYsareisostericwithtyrosine,presentarangeofphenolicpttTheEscherichiacoliribonucleotidereductase(RNR)catalyzesthepKa
6、saswellasalargerangeofoxidationpotentials(Table1).WithhconversionofallfournucleotidestotheircorrespondingtheproperchoiceofFnYandreactionpH,onecanchangethepuo2¢-deoxynucleotidesandconsistsoftwohomodimericsubunits:protonationstateofFnY356,itsoxidationpotentialorboth.Thus,rGR1and
7、R2(refs.13).R1isthehomodimericsubunitwherenucleotideFnYsareexcellentprobesforexaminingproton-coupledelectrongreductionoccurs.Itcontainsbindingsitesfornucleosidediphos-transfermechanismsinradicalpropagationandshouldbegen-nihphatesubstratesaswellasfordeoxynucleosidetriphosphatea
8、ndATPerallyusefulforstudyingproteinsthatrequirestableortransi
