University of Groningen Sec translocase in action Komarudin, Amalina Ghaisani
21
0
0
Hele tekst
(2) CHAPTER 3 SecYEG Mediates Translocation of 6HFUHWRU\3URWHLQ0RGLÀHGZLWK1RQ Polypeptide Segments "NBMJOB(IBJTBOJ,PNBSVEJO .JDLFM+)BOTFO #FO-'FSJOHB "SOPME+.%SJFTTFO to be submitted.
(3) Chapter 3. ABSTRACT In bacteria, signal sequence bearing secretory proteins (preproteins) are translocated BDSPTT UIF DZUPQMBTNJD NFNCSBOF CZ UIF 4FDUSBOTMPDBTF *O JUT NJOJNBM GPSN JU consists of a protein-conducting channel SecYEG, and the ATP-driven motor protein 4FD" )FSF XF IBWF FYBNJOFE UIF USBOTMPDBUJPO PG UIF TFDSFUPSZ QSPUFJO QSP0NQ" UIBUXBTDPOKVHBUFEUPQPMZFUIZMFOFHMZDPMTFHNFOUTXJUIEJGGFSFOUDPOUPVSMFOHUITBU DZTUFJOFTJOUSPEVDFEBUEJGGFSFOUQPTJUJPOT4VDIOPOQPMZQFQUJEFTFHNFOUTBSFSFBEJMZ USBOTMPDBUFECZ4FD:&(FYDFQUXIFOUIFJOUIFmSTUBNJOPBDJETPGUIFNBUVSFEPNBJO PGQSP0NQ"'VSUIFSNPSF TNBMMnVPSFTDFOUEZFTXFSFBMTPOPUUPMFSBUFEJOUIJTSFHJPO but the translocation defect could be restored by the PrlA4 mutant of SecY that exhibits BMPPTFOFESFRVJSFNFOUGPSDIBOOFMPQFOJOH5IFTFEBUBTVQQPSUUIFOPUJPOUIBUMPPQ formation between the signal sequence and the amino-terminal mature region of the secretory protein is critical for initiation of translocation, but that once translocation has CFFOJOJUJBUFE UIFUSBOTMPDBTFJTSBUIFSUPMFSBOUUPQSPUFJONPEJmDBUJPOT. 54.
(4) 4FD:&(.FEJBUFT5SBOTMPDBUJPOPG4FDSFUPSZ1SPUFJO.PEJmFEXJUI/PO1PMZQFQUJEF4FHNFOUT. INTRODUCTION In bacteria, the Sec translocase is the main system for the translocation of proteins BDSPTTUIFDZUPQMBTNJDNFNCSBOF<>*UDPOTJTUTPGBQSPUFJODPOEVDUJOHDIBOOFMDBMMFE SecYEG [2], and SecA, a peripheral associated component that provides the energy for UIFUSBOTMPDBUJPOQSPDFTT<>.PTUTFDSFUPSZQSPUFJOTBSFUSBOTMPDBUFEQPTUUSBOTMBUJPOBMMZ BGUFSUIFJSDPNQMFUFTZOUIFTJTBUUIFSJCPTPNF*OUIJTQSPDFTT UIFQSPUFJOCPVOEFBSMZ EVSJOHCJPTZOUIFTJTCZNPMFDVMBSDIBQFSPOFTTVDIBT4FD# LFQUVOGPMEFE<> BOE UBSHFUFEUPUIF4FD"QSPUFJOBUUIFNFNCSBOF<>4FD"CJOETXJUIMPXBGmOJUZUPBDJEJD QIPTQIPMJQJETBUUIFNFNCSBOFTXIFSFJUJTQSJNFEGPSUIFIJHIBGmOJUZJOUFSBDUJPOXJUI UIF4FD:&(QPSF5IJTJOUVSO BDUJWBUFT4FD"GPS"%1"51FYDIBOHF XIJDIBMMPXTUIF initiation of translocation wherein a looped structure of the signal sequence and the early NBUVSFSFHJPOPGUIFQSFQSPUFJOJOTFSUJOUPUIFUSBOTMPDBUJPOQPSF<>*OUFSDBMBUJPOPGUIF signal sequence into the lateral gate of SecY results in the opening of a central aqueous QPSFUIBUBMMPXTUIFQBTTBHFPGVOGPMEFEQPMZQFQUJEFTBDSPTTUIFNFNCSBOF5IFQPSF JTLFQUTFBMFECZUIFDFOUSBMQPSFSJOHPGIZESPQIPCJDBNJOPBDJETUIBUBDUTMJLFBHBTLFU BSPVOEUIFUSBOTMPDBUJOHQPMZQFQUJEF%JSFDUJPOBMMZUPUSBOTMPDBUJPOJTQSPWJEFECZ4FD" which supports cycles of ATP binding and hydrolysis that drive the complete translocation PGUIFQSFQSPUFJO<>*OUIFQSFTFODFPGUIFQSPUPONPUJWFGPSDF BOEJOUIFBCTFODFPG a SecA association, large segments of the preprotein may be translocated in a process UIBUJTTVQQPSUFECZUIFBDDFTTPSZ4FD%'NFNCSBOFQSPUFJODPNQMFY<> The SecYEG pore conducts the passage of unfolded proteins, but appears rather nonTFMFDUJWFBTGBSBTQPMZQFQUJEFDPNQPTJUJPODPODFSOT-POHFSTUSFUDIFTPGIZESPQIPCJD amino acid residues may, however, insert laterally into the membrane rather than being USBOTMPDBUFECZBQSPDFTTUIBUJOWPMWFTLJOFUJDQBSUJUJPOJOHBOEUIBUJTEJDUBUFECZUIF BWFSBHFIZESPQIPCJDJUZPGUIFUSBOTMPDBUJOHSFHJPO'VSUIFSNPSF SFMBUJWFMZTIPSUTUSFUDIFT of positively charged amino acid residues inhibit translocation, long stretches of glycine SFTJEVFTBSFUPMFSBUFE<>5IFMBUUFSTVHHFTUTUIBUUSBOTMPDBUJPOJTEFQFOEFOUPONBJO DIBJOJOUFSBDUJPOTSBUIFSUIBOTJEFDIBJOJOUFSBDUJPOT*OBEEJUJPO UIF4FD:&(QPSFBMTP DPOEVDUTUIFUSBOTMPDBUJPOPGOPOQFQUJEFDPOTUJUVFOUTUIBUBSFDPOKVHBUFEUPBQSFDVSTPS QSPUFJOJODMVEJOHnVPSFTDFOUQSPCFTBOECVMLZDIFNJDBMDPNQPVOET< >1SFWJPVT TUVEJFTIBWFTIPXOUIBU4FD:&(BMMPXTUIFUSBOTMPDBUJPOPGQSP0NQ"BUUBDIFEUPBCVMLZ nVPSPQIPSFTBUDZTUFJOFTQSFTFOUJOUIFDBSCPYZMUFSNJOVT<>1SP0NQ"DPOKVHBUFEXJUI rigid spherical organic molecules attached to the carboxyl-terminus are also translocated CVUXJUIBTJ[FMJNJUPG¯<>*OHFOFSBM JUBQQFBSTUIBUUIF4FD:&(QPSFJTSBUIFS VOTFMFDUJWF"TQFDJmDDBTFFYJTUTGPSUIF4FD"4FD:TZTUFNJOTPNFQBUIPHFOJD bacteria, that allow the translocation of precursor proteins that have been glycosylated in. 55. 3.
(5) Chapter 3. UIFDFMM<>5IFOPODBOPOJDBM4FD"4FD:TZTUFNSFRVJSFTBQQSPYJNBUFMZSFTJEVFT BEKBDFOUUPUIFTJHOBMQFQUJEF UIFTPDBMMFE"45EPNBJO GPSUIFUSBOTMPDBUJPOQSPDFTT <>)PXFWFS VOHMZDPTZMBUFEQSFDVSTPSQSPUFJOTBSFBMTPFYQPSUFEWJBUIJTTZTUFNXIJDI TVHHFTUJOHUIBUUIFHMZDPTZMBUJPOJTBOPUSFRVJSFNFOUGPSUSBOTMPDBUJPO<>"TXJUIUIF canonical Sec translocase, the precursor proteins needs to be partially unfolded prior to UIFUSBOTMPDBUJPOBOEBEEJUJPOBMMZ SFRVJSFETPNFBDDFTTPSZTFDSFUFEQSPUFJOTMJLF"TQ (BQ <> Previous studies showed the ATP dependent translocation into SecYEG proteoliposomes PG QSP0NQ" EFSJWBUJWFT XJUI CVMLZ OPOQPMZQFQUJEF NPEJmDBUJPO BU UIF $UFSNJOBM FOE<>)PXFWFS JUJTVOLOPXOUPXIBUFYUFOUUIF4FDUSBOTMPDPOUPMFSBUFTDIFNJDBM NPEJmDBUJPOT BU PUIFS QPTJUJPOT PG B QSFDVSTPS QSPUFJO 5P FYBNJOF UIF UPMFSBODF PG UIFUSBOTMPDPOGPSUSBOTMPDBUJPOPGDIFNJDBMMZNPEJmFEEFSJWBUJWFTPGQSP0NQ" CPUI nVPSPQIPSFTBTXFMMBTQPMZNFSJDIZESPQIJMJDPMJHPFUIZMFOFHMZDPMDIBJOTXFSFJOUSPEVDFE BUWBSJPVTQPTJUJPOTPGUIFQPMZQFQUJEFDIBJOWJBDIFNJDBMNPEJmDBUJPOPGVOJRVFDZTUFJOF SFTJEVFQPTJUJPOTGPDVTJOHPOUIFNBUVSF/UFSNJOBMSFHJPOPGQSP0NQ"5IFEBUBTIPXT UIBUUIFUSBOTMPDPOFYIJCJUTBSFNBSLBCMFQSPNJTDVJUZUPXBSETQPMZQFQUJEFTVCTUSBUFT XJUITVDIDIFNJDBMNPEJmDBUJPO CVUUIBUJUEPFTOPUUPMFSBUFNPEJmDBUJPOJOUIFNBUVSF /UFSNJOBMSFHJPO. MATERIAL AND METHODS Introduction of cysteine residue into proOmpA E. coli%)ЃXBTVTFEGPSNPMFDVMBSDMPOJOH1MBTNJEQ&5XBTVTFEBTUFNQMBUF in which endogenous cysteine residues at position 290 and 302 of proOmpA were TVCTUJUVUFEGPSBTFSJOF<>6TJOH2VJL$IBOHFTJUFEJSFDUFENVUBHFOFTJTNFUIPE B single cysteine residue was introduced into the Cysteine-less proOmpA at positions 8, 16 and 20 of the mature proOmpA yielding pAGK029 (Y8C, proOmpA 8C), pAGK031 4$ QSP0NQ"$ BOEQ"(, %$ QSP0NQ"$ Q&,XBTVTFEUP express a proOmpA variant with a single cysteine position at the extreme C-terminus PG UIF QSPUFJO <> "MM NVUBOUT BSF MJTUFE JO 5BCMF 5IF JOUSPEVDFE NVUBUJPOT XFSF DPOmSNFECZTFRVFODJOH. 56.
(6) 4FD:&(.FEJBUFT5SBOTMPDBUJPOPG4FDSFUPSZ1SPUFJO.PEJmFEXJUI/PO1PMZQFQUJEF4FHNFOUT. Table 2 | Strains and plasmids used in this study Strains/Plasmid. Description. Source. pET2345. Cysless proOmpA. [12]. pAGK029. proOmpA Y8C. This study. pAGK031. proOmpA S16C. This study. pAGK032. proOmpA D20C. This study. pET2348. proOmpA N33C. [12]. pEK204. proOmpA 326C. [17]. pET502. proOmpA C302S. [18]. pET2302. SecYEG. [19]. pET2306. PrlA4 mutant. [19]. E. coli %)Ѓ. TVQ& åMBD6 åMBD;@. ITE3 SFD" FOE" [20] HZS" UIJSFM" -. E. coli SF100. F åMBD9 HBM& HBM, UIJ SQT- TUS" åQIP"(pvuII), DompT. [21]. E. coli MM52. F-, åMBDU169, araD139, rpsL, thi, relA, ptsF25, deoC1, secA51. [22]. 3. 3XULÀFDWLRQRISUR2PS$ E. coli ..XBTVTFEUPFYQSFTTUIFTFUPGQSP0NQ"WBSJBOUT5SBOTGPSNFEE. coli ..DFMMTXFSFHSPXOPWFSOJHIUJO-#XJUIHMVDPTFBOEBNQJDJMMJO NHN- BU$'SPNUIJTDVMUVSF N-XBTVTFEUPJOPDVMBUFN-PG-#TVQQMFNFOUFEXJUI HMVDPTFBOEBNQJDJMMJO NHN- BOEHSPXUIXBTDPOUJOVFEBU$GPSIPVST To reduce the remaining concentration of glucose, the culture was diluted with preXBSNFEN-PG-# $ TVQQMFNFOUFEXJUIBNQJDJMMJO NHN- "GUFSNJOPG growth at 37 ºC, high-level expression of the ompA gene was induced by the addition PGN.JTPQSPQZMUIJPЄ%HBMBDUPQZSBOPTJEF *15( GPSIPVSTBOEUIFDFMMTXFSF IBSWFTUFE XBTIFEXJUIN.)&1&4,0)Q)BOESFTVTQFOEFEJOBCPVUN- PGUIFTBNFCVGGFS 5IF DFMMT XFSF EJTSVQUFE BOE TVQQMFNFOUFE XJUI N. /B&%5" BOE N. QIFOZMNFUIZMTVMGPOZMnVPSJEF 1.4' *ODMVTJPOCPEJFTBOEDFMMEFCSJTXFSFDPMMFDUFE by low-speed centrifugation (2000 g, 10 min), and dissolved in 5 mL of 8 M urea, 20 N.)&1&4,0)Q) GPMMPXFECZIJHITQFFEDFOUSJGVHBUJPO H NJO UP SFNPWFUIFOPOTPMVCMFNBUFSJBM5IFTVQFSOBUBOUXBTMPBEFEPOB)J5SBQ24FQIBSPTF DPMVNO (&)FBMUIDBSF FRVJMJCSBUFEXJUI.VSFB N.)&1&4,0) Q) BOE UIFDPMVNOXBTXBTIFEXJUIUIFTBNFCVGGFS5IFDPOUBNJOBUJOHQSPUFJOTSFNBJOCPVOE UPUIFDPMVNO XIFSFBTQSP0NQ"FMVUFTXJUIUIFOPOCPVOEQSPUFJOGSBDUJPO. 57.
(7) Chapter 3. Labeling of proOmpA mutants 1SP0NQ"MBCFMJOHXBTDPOEVDUFEVTJOHTPMJEQIBTFMBCFMJOHNFUIPEBTEFTDSJCFE<> #SJFnZ UIFQSP0NQ"NVUBOUTXFSFSFEVDFEXJUIN.EJUIJPUISFJUPM %55 GPSNJOVUFT BOEQSFDJQJUBUFEXJUIUSJDIMPSPBDFUJDBDJE 5$" %55XBTSFNPWFECZBDFOUSJGVHBUJPO TUFQ &QQFOEPSG NJOVUFT 5IFQSPUFJOQFMMFUXBTXBTIFEXJUIJDFDPMEBDFUPOFBOE EJTTPMWFEJO.VSFB N.)&1&4,0)Q)DPOUBJOJOHGPMENPMBSFYDFTTPGFJUIFS nVPSFDFJONBMFJNJEF 'NBM NBMFJNJEF1&(1000 (malPEG1000) or maleimide-PEG3000 (malPEG3000 BOEJODVCBUFEBUSPPNUFNQFSBUVSFGPSIPVST5IFSFBDUJPOXBTTUPQQFE by adding 5 mM of DTT and the unreacted labeled proOmpA was collected by TCA QSFDJQJUBUJPO5IFMBCFMFEQSPUFJOXBTSFTVTQFOEFEJO.VSFB N.)&1&4,0) Q)BOEBOBMZ[FEXJUI4%41"(&5IFnVPSFTDFODFPGQSP0NQ"nVPFTFDFJO NBMFJNJEF QSP0NQ"'NBM XBTWJTVBMJ[FEVTJOHB'VKJmMN-"4JNBHFBOBMZ[FS 8JUINBM1&(1000 and malPEG3000 UIFMBCFMJOHPGQSP0NQ"XBTDPOmSNFECZBTMPXFS migration on SDS-PAGE of the labeled protein in comparison to the unlabeled protein BOETUBJOJOHVTJOHCBSJVNJPEJOF<>1SPUFJODPODFOUSBUJPOTXFSFEFUFSNJOFEVTJOHUIF #JP3BE%$QSPUFJOBTTBZXJUICPWJOFTFSVNBMCVNJOBTBTUBOEBSE In vitro translocation assay Translocation of the set of labeled proOmpA into SecYEG or PrlA4EG proteoliposomes XBTBTTBZFECZUIFBDDFTTJCJMJUZUPFYUFSOBMMZBEEFEQSPUFJOBTF,<>5IFUSBOTMPDBUJPO SFBDUJPOXBTQFSGPSNFEJOUSBOTMPDBUJPOCVGGFS N.)&1&4,0) Q) N.,$M NHN-CPWJOFTFSVNBMCVNJO N.%55 N..H$M2) with 10 mM phosphocreatine, HN-DSFBUJOFLJOBTF HN-PG4FD# O.PG4FD" -4FD:&(PS1SM"&( QSPUFPMJQPTPNFT BOEO.PGMBCFMFEQSP0NQ"5SBOTMPDBUJPOTUBSUFECZUIFBEEJUJPO PGN."515IFSFBDUJPOXBTJODVCBUFEBU$GPSNJOVUFTBOEUFSNJOBUFECZ DIJMMJOHUIFTBNQMFTPOJDFGPMMPXJOHUIFQSPUFJOBTF,USFBUNFOU5IFUSBOTMPDBUFEPG proOmpA-Fmal and proOmpA-malPEG were analyzed by SDS-PAGE, followed by in HFMWJTVBMJ[BUJPOVTJOH'VKJ*NBHFS<>BOEJNNVOPCMPUBHBJOTU0NQ" SFTQFDUJWFMZ<> "TJOUFSOBMTUBOEBSE -PGUIFSFBDUJPONJYUVSFJTNJYFEXJUIY4%4TBNQMFCVGGFS. RESULTS Importance of the proOmpA mature N-terminus for translocation 5PFYBNJOFUIFUPMFSBODFPGUIF4FDUSBOTMPDPOGPSDIFNJDBMNPEJmDBUJPOTPGTVCTUSBUF precursor proteins, unique cysteine positions were introduced into proOmpA for TVCTFRVFOUMZ NBMFJNJEF EJSFDUFE EFSJWBUJ[BUJPO 1SP0NQ" DPOTJTUT PG UXP EPNBJOT an N-terminal hydrophobic beta barrel embedded in the membrane and an exposed $UFSNJOBMSFHJPOUIBUSFTJEFTJOUIFQFSJQMBTN 'JH" <>6OJRVFTJOHMFDZTUFJOF. 58.
(8) 4FD:&(.FEJBUFT5SBOTMPDBUJPOPG4FDSFUPSZ1SPUFJO.PEJmFEXJUI/PO1PMZQFQUJEF4FHNFOUT. residues were introduced at positions 4, 8, 16, 20, 33, using the cysteine-less proOmpA BTBUFNQMBUF 'JH" 1SP0NQ"XJUIUIFOBUJWFDZTUFJOFQPTJUJPOBOEBUUIF FYUSFNF$UFSNJOVTXFSFVTFEBTDPOUSPMT"MMQSP0NQ"NVUBOUTXFSFPWFSFYQSFTTFE in E. coli MM52, a temperature sensitive SecA strain that harbors a lethal mutation of 4FD""UUIFTVCNJTTJWFUFNQFSBUVSFPG$ UIJTTUSBJOTIPXTBNBKPSTFDSFUJPOEFGFDU DBVTJOHUIFBDDVNVMBUJPOPGQSP0NQ"BTJODMVTJPOCPEJFT<>5IFJODMVTJPOCPEJFT XFSF FYUSBDUFE XJUI VSFB BOE QSP0NQ" NVUBOUT XFSF QVSJmFE XJUI BOJPO FYDIBOHF DISPNBUPHSBQIZ 'JH# 5IFQVSJmFEQSP0NQ"NVUBOUTJOVSFBXFSFMBCFMFEXJUI UIFnVPSFTDFOUEZFnVPSFTDFJONBMFJNJEF 'NBM 'JH$ VTJOHBTPMJEQIBTFMBCFMJOH QSPUPDPM<>. 3. Fig. 1. Fluorescent labeling of proOmpA " 4DIFNFPGQSP0NQ"TJOHMFDZTUFJOFNVUBOU8JME UZQFQSP0NQ"DPOUBJOTUXPDZTUFJOFSFTJEVFTBUUIF$UFSNJOVTPGUIFQPTJUJPOBOE # 1VSJmFETJOHMFDZTUFJOFQSP0NQ"NVUBOUTMBCFMFEXJUI'MVPSFTDFJONBMFJNJEF 'NBM $ 5IF DPPNBTJFTUBJOFEPGTJOHMFDZTUFJOFQSP0NQ"'NBMBTBDPOUSPMTIPXJOHUIFMBCFMJOHFGmDJFODZ. In line with previous data [12,17], translocation was observed when the Fmal was DPOKVHBUFE BU QPTJUJPO BOE PG QSP0NQ" 'JH CMBDL CBS 8JUI UIF 'NBM labeled position 33C, translocation was slightly inhibited, but labeling at position 4, 8, BOEESBNBUJDBMMZSFEVDFEUIFFGmDJFODZPGQSP0NQ"USBOTMPDBUJPO 'JH XIJMF BMMVOMBCFMFEQSP0NQ"TJOHMFDZTUFJOFNVUBOUTXFSFFGmDJFOUMZUSBOTMPDBUFECZ4FD:&( QSPUFPMJQPTPNFT EBUBOPUTIPXO 4JODFUIFDSJUJDBMQPTJUJPOTBSFJOUIFFYUSFNFNBUVSF N-terminus, labeling could potential interfere with loop formation of the signal sequence BOENBUVSF/UFSNJOVTBOEUIFSFCZQSFWFOUDIBOOFMPQFOJOH5IFSFGPSF USBOTMPDBUJPO XBTBMTPUFTUFEXJUI1SM"&(QSPUFPMJQPTPNFT5IF1SM"NVUBOUPG4FD:&(JTBTUSPOH suppressor of signal sequence mutations, and such mutations are thought to destabilize. 59.
(9) Chapter 3. the translocation pore thereby even allowing the translocation of signal sequenceMFTTNVUBOUT< >*NQPSUBOUMZ UIFQSP0NQ"'NBMEFSJWBUJWFTTIPXFETJHOJmDBOUMZ FOIBODFE USBOTMPDBUJPO BDUJWJUJFT XJUI 1SM"&( QSPUFPMJQPTPNFT 'JH XIJUF CBS 5IFTFmOEJOHTVHHFTUTUIBUQSPQFSBMJHONFOUPGUIFmSTUBNJOPBDJETPGUIFNBUVSF /UFSNJOVTPGQSP0NQ"JTDSJUJDBMGPSDIBOOFMPQFOJOHBOEUSBOTMPDBUJPO. Fig. 2 The in vitro translocation assay of proOmpA-Fmal labeled5IF'NBMBUUBDINFOUBUUIF $UFSNJOVTEJEOPUJOUFSGFSFUIFUSBOTMPDBUJPOBDUJWJUZ$POKVHBUJOH'NBMUPBDZTUFJOFBUUIFmSTU BNJOPBDJEPGUIFNBUVSFEPNBJOPGQSP0NQ"SFEVDFEUIFUSBOTMPDBUJPOFGmDJFODZXJUI4FD:&( QSPUFPMJQPTPNFT CMBDLCBS NFBOXIJMFUIFSFTUPSBUJPOXBTPCTFSWFEXJUI1SM"&(DPNQMFYQSPUFPMJQPTPNFT XIJUFCBS . 7UDQVORFDWLRQRISUR2PS$GHULYDWLYHVPRGLÀHGZLWKSRO\PHULFSRO\HWK\OHQH glycol chains To gain more insight in the promiscuity of translocon for non-polypeptide entities DPOKVHBUFEUPQSFDVSTPSQSPUFJOT UIFTJOHMFDZTUFJOFNVUBOUTPGQSP0NQ"XFSFBMTP MBCFMFEXJUIOPOOBUVSBMQPMZNFSTPGFUIZMFOFHMZDPMBUUBDIFEWJBBNBMFJNJEFMJOLFS The inert and hydrophilic properties of the polyethyleneglycol is important as to QSFWFOUVODPOUSPMMFEQSPUFJOJOUFSBDUJPOT5PWBSZUIFTJ[FPGUIFNPMFDVMFT NBMFJNJEF polyethyleneglycol1000 (malPEG1000) and maleimide polyethyleneglycol3000 (malPEG3000) XFSF TZOUIFTJ[FE XJUI BOE SFQFBUJOH VOJUT SFTQFDUJWFMZ 'JH " "MM PG UIF QSP0NQ"NVUBOUTXFSFMBCFMFEBOETVCKFDUFEUP4%41"(&BOBMZTJTBOEDPPNBTJF TUBJOJOH*OBEEJUJPO CBSJVNJPEJOFTUBJOJOHDPOmSNFEUIFBUUBDINFOUPGNBMFJNJEF1&( 'JH# BTJUTQFDJmDBMMZGPSNTBCBSJVNJPEJEF1&(DPNQMFY<>. 60.
(10) 4FD:&(.FEJBUFT5SBOTMPDBUJPOPG4FDSFUPSZ1SPUFJO.PEJmFEXJUI/PO1PMZQFQUJEF4FHNFOUT. "TTIPXOJO'JH# MBCFMJOHPGDZTUFJOFNVUBOUTPGQSP0NQ"XJUINPOP1&(1000 causes BTIJGUPGUIFNPCJMJUZPO4%41"(&5IFNPMFDVMBSTJ[FPGUIFDPOKVHBUFJTBSPVOE L%BCVUUIFDIBSHFPGUIFQSPUFJOJUTFMGJTOPUBGGFDUFE"OFYUSBCBOEXJUIBNPMFDVMBS weight corresponding to proOmpA-di-PEG1000 XBT BMTP PCTFSWFE 5IJT VQQFS CBOE SFTVMUGSPNUIFMBCFMJOHPGDPQVSJmFEFOEPHFOPVTQSP0NQ"UIBUIBTUXPDZTUFJOFT BUQPTJUJPOTBOE1SP0NQ"NPOPNBM1&(1000XBTUIFNBKPSQSPEVDUGPSNFE BGUFS MBCFMMJOH ö 1SP0NQ"NBM1&(3000 migrated even slower than proOmpAmalPEG1000"TNBM1&(3000JTSFMBUJWFMZCVMLJFSUIBONBM1&(1000, the degree of labeling with malPEG3000XBTOPUBTFGmDJFOUBTXJUINBM1&(1000, and about half of the proOmpA SFNBJOFEVOSFBDUFE 'JH# 5IFVOMBCFMFEQSP0NQ"XBTVTFEBTBOJOUFSOBMDPOUSPM UPEFUFSNJOFJGUIFNPEJmFETVCTUSBUFCMPDLFEUSBOTMPDBUJPO. 3. Fig. 3 Conjugation of proOmpA with the maleimide polyethyleneglycol. (A) Overview of the TUSVDUVSFTPGNBMFNJEFQPMZFUIZMFOFHMZDPM NBM1&( VTFEUPMBCFMQSP0NQ"DZTUFJOFNVUBOUT # proOmpA 8C as the representative mutant labeled with malPEG1000 (lane 2) and malPEG3000 (lane XFSFTFQBSBUFECZ4%41"(&BOETUBJOFEXJUI$PPNBTTJF#SJMMJBOU#MVF $ 5IFJPEJOF stained of proOmpA-malPEG1000 (lane 5) and proOmpA-malPEG3000 MBOF 1PTJUJPOTPGNPMFDVMBS NBTTNBSLFST JOL%B BSFJOEJDBUFE. 61.
(11) Chapter 3. The set of proOmpA mutants labeled with the different malPEG structures were assayed GPSUSBOTMPDBUJPO VTJOHJNNVOPCMPUUJOHBHBJOTU0NQ"UPEFUFDUUIFUSBOTMPDBUFETVCTUSBUF The immunostaining visualizes only the completed and very late stages of proOmpA translocation as the polyclonal OmpA antibody recognizes only the extreme C-terminus PG0NQ"<>*OUIFBCTFODFPG"51 OPUSBOTMPDBUJPOPDDVSTBOEUIFNBM1&(1000 labeled QSP0NQ"JTDPNQMFUFMZEJHFTUFECZQSPUFJOBTF, 'JH *OUFSFTUJOHMZ UIFNBM1&(1000 labeled proOmpA could be translocated across the SecYEG channel when the labeling XBTBUQPTJUJPOBOEPOXBSETVQUPUIF$UFSNJOVT 'JH MBOFGPS4FD:&( 4USJLJOHMZ USBOTMPDBUJPOXBTTFWFSFMZJNQBJSFEXIFOUIFNBM1&(1000 was attached to QPTJUJPOBOE 'JH MBOFGPS4FD:&( 6TJOH1SM"&(QSPUFPMJQPTPNFT UIF USBOTMPDBUJPOFGmDJFODZPGQSP0NQ"MBCFMFEXJUINBM1&(1000 at positions 16, 20 and 33 XBTJODSFBTFEDPNQBSFEUP4FD:&(QSPUFPMJQPTPNFT 'JH DPNQBSFMBOFGPS 4FD:&(BOE1SM"&( SFTQFDUJWFMZ 4VSQSJTJOHMZ UIFUSBOTMPDBUJPOPGQSP0NQ"MBCFMFE BU UIF /UFSNJOBM QPTJUJPOT BOE 'JH DPNQBSF MBOF MBOF GPS 4FD:&( BOE 1SM"&( SFTQFDUJWFMZ DPVMEOPUCFSFTUPSFE. Fig. 4 Translocation of unlabeled and labeled proOmpA 326C into SecYEG proteoliposomes (A) Unlabeled proOmpA, proOmpA-Fmal and proOmpA-malPEG1000XFSFVTFEJOUIFBTTBZ-BOF BSFUIFPGUPUBMUIFQSP0NQ"EFSJWBUJWFJOPOFSFBDUJPO5SBOTMPDBUJPOQSP0NQ"CZUIF indicated ATP was added (lane 10, 12 and 14) and not added to the reaction (lane 9, 11 and 13), XIJMFJOMBOF4FD"XBTPNJUUFEGSPNUIFSFBDUJPOQSP0NQ"NBM1&(XBTBCMFUPUSBOTMPDBUF 4FD:&(QSPUFPMJQPTPNFTOJDFMZBTUIFQSP0NQ"'NBMBOEVOMBCFMFEQSP0NQ" # 5SBOTMPDBtion proOmpA-malPEG1000 and proOmpA malPEG3000 4FD:&(QSPUFPMJQPTPNFT-BOFBOE BSFUIFDPOUSPMTBNQMFTPGUIFSFBDUJPOGPSQSP0NQ"NBM1&(1000 and proOmpA-malPEG3000, SFTQFDUJWFMZ*OMBOFBOE UIF"51XBTPNJUUFEJOUIFSFBDUJPO5IFBTTBZGPMMPXFECZJNNVOPTUBJOJOHVTJOHBQPMZDMPOBMBOUJCPEZBHBJOTU0NQ". 62.
(12) 4FD:&(.FEJBUFT5SBOTMPDBUJPOPG4FDSFUPSZ1SPUFJO.PEJmFEXJUI/PO1PMZQFQUJEF4FHNFOUT. Fig. 5. Translocation of proOmpA-malPEG1000 into proteoliposomes of SecYEG and PrlA4EG complex.-BOFJTUIFPGUIFQSP0NQ"VTFEJOUIFUSBOTMPDBUJPOSFBDUJPOQSP0NpA-malPEG1000 was able to translocate SecYEG and PrlA4EG proteoliposomes (lane 10-14), but not with proOmpA-4C-malPEG1000 and proOmpA-8C-malPEG1000 MBOF . 5IFTBNFUSFOEXBTPCTFSWFEGPSQSP0NQ"EFSJWBUJWFTDPOKVHBUFEXJUINBM1&(3000 Interestingly, proOmpA-malPEG3000NPEJmFEBUQPTJUJPO BOE 'JH MBOF NJHSBUFETMJHIUMZEJGGFSFOUUIBOXIFODPOKVHBUFEBU$BOE$ 'JH MBOF 4FD:&(XBTBMTPBCMFUPUSBOTMPDBUFUIFQSP0NQ"MBCFMFEXJUINBM1&(3000 at the MBUUFSOBUJWFDZTUFJOFQPTJUJPOT 'JH MBOF *NQPSUBOUMZ UIFVOMBCFMFEQSP0NQ" XBTFGmDJFOUMZUSBOTMPDBUFEJOBMMSFBDUJPOTPGUSBOTMPDBUJPO FWFOBUQPTJUJPOTBOE 'JH MBOFBOE'JH MBOF XIJDIEFNPOTUSBUFTUIBUUIFQSP0NQ"EFSJWBUJWFT EFGFDUJWFJOUSBOTMPDBUJPOEPOPUCMPDLUIFUSBOTMPDBUJPODIBOOFM5IFTFEBUBEFNPOTUSBUF BIJHIQSPNJTDVJUZPGUIFUSBOTMPDPOGPSNPEJmFEQSP0NQ"TVCTUSBUFT FYDFQUXIFOUIF NPEJmDBUJPOTPDDVSBUUIFFYUSFNFNBUVSF/UFSNJOVT. Fig. 6. Translocation of proOmpA-malPEG3000 into proteoliposomes of SecYEG and PrlA4EG complex4FD:&(BMMPXTUIFUSBOTMPDBUJPOPGQSP0NQ"NBM1&(3000 (lane 10-14), also proOmpA di-malPEG3000 MBOF FYDFQUBUUIFQPTJUJPOPG$BOE$ MBOF . 63. 3.
(13) Chapter 3. DISCUSSION Protein translocation is a stepwise process in which each turnover is responsible for transport of a distinct length of polypeptide [18,30], whereas other regions may USBOTMPDBUFUISPVHIBTMJEJOHNPMFDVMBSSBUDIFUNFDIBOJTN5SBOTMPDBUJPOJTJOJUJBUFECZ the recognition of the N-terminal hydrophobic signal sequence at the SecYEG translocon <>*OUIFQSFTFOUXPSL XFHFOFSBUFEBTFUPGTJOHMFDZTUFJOFQSP0NQ"NVUBOUTMBCFMFE XJUInVPSFTDFOUQSPCFBOEIZESPQIJMJDPMJHPFUIZMFOFHMZDPMQPMZNFSTPGEJGGFSFOUDPOUPVS MFOHUIT SFTQFDUJWFMZ0VSEBUBTIPXTUIBUXIFO'MVPSFTDFJONBMFJNJEFJTBUUBDIFEUPB TQFDJmDDZTUFJOFQPTJUJPOXJUIJOUIFmSTUBNJOPBDJETPGUIFQSP0NQ"NBUVSFEPNBJO UIFUSBOTMPDBUJPOJTBCPMJTIFE)PXFWFS UIJTEFGFDUJWFUSBOTMPDBUJPODPVMECFSFTUPSFE with proteoliposomes reconstituted with the PrlA4 mutant of SecY that exhibits a loosed SFRVJSFNFOUGPSDIBOOFMPQFOJOH<>5IJTSFTVMUTVHHFTUTUIBUnVPSPQIPSFTBUUIFNBUVSF /UFSNJOVTJOUFSGFSFXJUIDIBOOFMPQFOJOH BOEMJLFMZDBOCFBUUSJCVUFEUPJOUFSGFSFODF with the formation of a hairpin of this region with the signal sequence that is critical for UIFUSBOTMPDBUJPOQSPDFTT 5IF4FD:&(QPSFJTIJHIMZnFYJCMFBTJUJTBCMFUPUSBOTMPDBUFQSFQSPUFJOTDSPTTMJOLFEXJUI BOPOQFQUJEFDPOTUJUVFOUT< >)FSFXFTIPXUIBUQSP0NQ"EFSJWBUJWFTMBCFMFEXJUI malPEG1000 and malPEG3000, respectively, remain translocation-competent in vitro 'JH .PSFPWFS FWFOUSBOTMPDBUJPOPGQSP0NQ"XBTPCTFSWFEUIBUXBTMBCFMFEBUUIFUXP endogenous cysteine positions with malPEG3000 'JH *NQPSUBOUMZ USBOTMPDBUJPOXBT POMZPCTFSWFEXIFOUIFMBCFMJOHPDDVSSFEBUDBSCPYZMUFSNJOBMDZTUFJOFQPTJUJPOT-JLFMZ the unstructured PEG molecule attached to proOmpA was able to cross the translocation QPSFWJBBTMJEJOHNPWFNFOU<> )PXFWFS XIFOUIFQPMZNFSTXFSFJOUSPEVDFEBUDZTUFJOFQPTJUJPOPS USBOTMPDBUJPO XBTBCPMJTIFEBOEUIJTQSPDFTTDPVMEOPUCFSFTUPSFECZUIF1SM"NVUBOUPG4FD: 'JH "SFDFOUTUVEZCZ'FTTM FUBM<>SFQPSUFEUIBUUIF4FD:&(QMVHJTBCMFUPCF EJTQMBDFECZUIFTJHOBMTFRVFODFPOMZ CVUJUJTOPUTVGmDJFOUFOPVHIUPGVMMZPQFOUIF DIBOOFM-JLFMZ UIFBEKBDFOUNBUVSFQPMZQFQUJEFDIBJOUPHFUIFSXJUITJHOBMTFRVFODF BSFSFRVJSFEGPSDIBOOFMPQFOJOH< >0VSEBUBTVHHFTUTUIBUUIFBUUBDINFOUPG UIFCVMLZQPMZFUIZMFOFHMZDPMNPJFUJFTBUUIFFYUSFNFNBUVSF/UFSNJOVTPGQSP0NQ" inhibit translocation through steric hindrance that perturbs of the opening of the SecYEG DIBOOFM 5PTVNNBSJ[F PVSEBUBEFNPOTUSBUFUIBU4FD:&(JTJOUPMFSBOUUPDIFNJDBMNPEJmDBUJPO PG QSP0NQ" XIFO TVDI NPEJmDBUJPO PDDVS BU UIF FYUSFNF NBUVSF /UFSNJOVT *U JT. 64.
(14) 4FD:&(.FEJBUFT5SBOTMPDBUJPOPG4FDSFUPSZ1SPUFJO.PEJmFEXJUI/PO1PMZQFQUJEF4FHNFOUT. concluded that correct alignment of the N-terminal mature region of the precursor protein with the signal peptide plays an critical a role in the initiation of translocation CVUPODFUSBOTMPDBUJPOIBTTUBSUFE CVMLZDIFNJDBMTBUUBDIFEUPUIFQSFDVSTPSQSPUFJO DPUSBOTMPDBUFVOSFTUSJDUFE. 3. 65.
(15) Chapter 3. REFERENCES %SJFTTFO"+./PVXFO/ 1SPUFJO5SBOTMPDBUJPO"DSPTTUIF#BDUFSJBM$ZUPQMBTNJD .FNCSBOFAnnu. Rev. Biochem. 77 o #JFLFS ,- 1IJMMJQT (+ 4JMIBWZ 5+ 5IF sec and prl genes of Escherichia coli J. Bioenerg. Biomembr. 22 o &DPOPNPV"8JDLOFS8 4FD"QSPNPUFTQSFQSPUFJOUSBOTMPDBUJPOCZVOEFSHPJOH "51ESJWFODZDMFTPGNFNCSBOFJOTFSUJPOBOEEFJOTFSUJPOCell 78 o 'FLLFT1 WBOEFS%PFT$%SJFTTFO"+ 5IFNPMFDVMBSDIBQFSPOF4FD#JTSFMFBTFE GSPNUIFDBSCPYZUFSNJOVTPG4FD"EVSJOHJOJUJBUJPOPGQSFDVSTPSQSPUFJOUSBOTMPDBUJPOEMBO J. 16 o )BSUM'6 -FDLFS4 4DIJFCFM& )FOESJDL+18JDLOFS8 5IFCJOEJOHDBTDBEFPG SecB to SecA to SecY E mediates preprotein targeting to the E. coliQMBTNBNFNCSBOFCell 63 o #SVOEBHF- )FOESJDL+1 4DIJFCFM& %SJFTTFO"+8JDLOFS8 5IFQVSJmFEE. coli JOUFHSBMNFNCSBOFQSPUFJO4FD:&JTTVGmDJFOUGPSSFDPOTUJUVUJPOPG4FD"EFQFOEFOUQSFDVSTPS QSPUFJOUSBOTMPDBUJPOCell 62 o ;JNNFS+ /BN:3BQPQPSU5" 4USVDUVSFPGBDPNQMFYPGUIF"51BTF4FD"BOEUIF QSPUFJOUSBOTMPDBUJPODIBOOFMNature 455 o 5TVLB[BLJ5/VSFLJ0 5IFNFDIBOJTNPGQSPUFJOFYQPSUFOIBODFNFOUCZUIF4FD%' NFNCSBOFDPNQPOFOUBiophysics (Oxf). 7 o /PVXFO/ #FSSFMLBNQ(%SJFTTFO"+. $IBSHFEBNJOPBDJETJOBQSFQSPUFJOJOIJCJU 4FD"EFQFOEFOUQSPUFJOUSBOTMPDBUJPOJ. Mol. Biol. 386 o ,BUP..J[VTIJNB4 5SBOTMPDBUJPOPGDPOKVHBUFEQSFTFDSFUPSZQSPUFJOTQPTTFTTJOH an internal non-peptide domain into everted membrane vesicles in Escherichia coliJ. Biol. Chem. 268 o #POBSEJ' )BM[B& 8BMLP. %V1MFTTJT' /PVXFO/ 'FSJOHB#-%SJFTTFO"+. 1SPCJOHUIF4FD:&(USBOTMPDBUJPOQPSFTJ[FXJUIQSFQSPUFJOTDPOKVHBUFEXJUITJ[BCMFSJHJE TQIFSJDBMNPMFDVMFTProc. Natl. Acad. Sci. U. S. A. 108 o ,FZ[FS+%F %PFT$7BO%FS%SJFTTFO"+. ,JOFUJD"OBMZTJTPGUIF5SBOTMPDBUJPOPG Fluorescent Precursor Proteins into Escherichia coli.FNCSBOF7FTJDMFT 277 o 1SBCVEJBOTZBI*%SJFTTFO"+. 5IF$BOPOJDBMBOE"DDFTTPSZ4FD4ZTUFNPG(SBN QPTJUJWF#BDUFSJB*OQQo4QSJOHFS $IBN #FOTJOH #" 4VMMBN 1. 5SBOTQPSU PG QSFQSPUFJOT CZ UIF BDDFTTPSZ 4FD TZTUFN SFRVJSFTBTQFDJmDEPNBJOBEKBDFOUUPUIFTJHOBMQFQUJEFJ. Bacteriol. 192 o #FOTJOH#" 5BLBNBUTV%4VMMBN1. %FUFSNJOBOUTPGUIFTUSFQUPDPDDBMTVSGBDF HMZDPQSPUFJO(TQ#UIBUGBDJMJUBUFFYQPSUCZUIFBDDFTTPSZ4FDTZTUFNMol. Microbiol. 58, o #FOTJOH#"4VMMBN1. "OBDDFTTPSZTFDMPDVTPGStreptococcus gordonii is required GPSFYQPSUPGUIFTVSGBDFQSPUFJO(TQ#BOEGPSOPSNBMMFWFMTPGCJOEJOHUPIVNBOQMBUFMFUT Mol. Microbiol. 44 o. 66.
(16) 4FD:&(.FEJBUFT5SBOTMPDBUJPOPG4FDSFUPSZ1SPUFJO.PEJmFEXJUI/PO1PMZQFQUJEF4FHNFOUT. 5PNLJFXJD[% /PVXFO/ -FFVXFO37BO 5BOT4%SJFTTFO"+. 4FD"4VQQPSUTB $POTUBOU3BUFPG1SFQSPUFJO5SBOTMPDBUJPO Ƒ281 o 7BO%FS8PML+18 %F8JU+(%SJFTTFO"+. 5IFDBUBMZUJDDZDMFPGUIFEscherichia coli4FD""51BTFDPNQSJTFTUXPEJTUJODUQSFQSPUFJOUSBOTMPDBUJPOFWFOUTEMBO J. 16, 7297– EF,FZ[FS+ WBOEFS%PFT$ 4XBWJOH+%SJFTTFO"+. 5IF':NVUBUJPOPG1SM" UFNQFSTUIFTJHOBMTFRVFODFTVQQSFTTPSQIFOPUZQFCZSFEVDJOHUIF4FD"CJOEJOHBGmOJUZ FEBS Lett. 510 o )BOBIBO% 4UVEJFTPOUSBOTGPSNBUJPOPGEscherichia coliXJUIQMBTNJETJ. Mol. Biol. 166 o #BOFZY ' (FPSHJPV ( In vivo degradation of secreted fusion proteins by the Escherichia coliPVUFSNFNCSBOFQSPUFBTF0NQ5J. Bacteriol. 172 o 0MJWFS%##FDLXJUI+ 3FHVMBUJPOPGBNFNCSBOFDPNQPOFOUSFRVJSFEGPSQSPUFJO secretion in Escherichia coliCell 30 o ,JN: )P40 (BTTNBO/3 ,PSMBOO: -BOEPSG&7 $PMMBSU'38FJTT4 &GmDJFOU TJUFTQFDJmDMBCFMJOHPGQSPUFJOTWJBDZTUFJOFTBioconjug. Chem. 19 o 'FF$+7BO"MTUJOF+. 1VSJmDBUJPOPGQFHZMBUFEQSPUFJOTMethods Biochem. Anal. 54 o ,VTUFST* WBOEFO#PHBBSU( EF8JU+ ,SBTOJLPW7 1PPMNBO#%SJFTTFO" 1VSJmDBUJPO BOEGVODUJPOBMSFDPOTUJUVUJPOPGUIFCBDUFSJBMQSPUFJOUSBOTMPDBUJPOQPSF UIF4FD:&(DPNQMFY Methods Mol. Biol. 619 o 3JFE( ,PFCOJL3 )JOEFOOBDI* .VUTDIMFS#)FOOJOH6 .FNCSBOFUPQPMPHZBOE assembly of the outer membrane protein OmpA of Escherichia coli,Mol. Gen. Genet. 243 o 7BOEFS%PFT$ %F,FZ[FS+ 7BOEFS-BBO.%SJFTTFO"+. 3FDPOTUJUVUJPOPG 1VSJmFE#BDUFSJBM1SFQSPUFJO5SBOTMPDBTFJO-JQPTPNFTMethods Enzymol. 372 o WBOEFS8PML+18 'FLLFT1 #PPSTNB" )VJF+- 4JMIBWZ5+%SJFTTFO"+ 1SM" QSFWFOUTUIFSFKFDUJPOPGTJHOBMTFRVFODFEFGFDUJWFQSFQSPUFJOTCZTUBCJMJ[JOHUIF4FD"4FD: JOUFSBDUJPOEVSJOHUIFJOJUJBUJPOPGUSBOTMPDBUJPOEMBO J. 17 o Kurfürst MM (1992) Detection and molecular weight determination of polyethylene glycolNPEJmFEIJSVEJOCZTUBJOJOHBGUFSTPEJVNEPEFDZMTVMGBUFQPMZBDSZMBNJEFHFMFMFDUSPQIPSFTJT Anal. Biochem. 200 o 4DIJFCFM& %SJFTTFO"+ )BSUM'68JDLOFS8 %FMUBNV)
(17) BOE"51GVODUJPOBU EJGGFSFOUTUFQTPGUIFDBUBMZUJDDZDMFPGQSFQSPUFJOUSBOTMPDBTFCell 64 o 'FLLFT1%SJFTTFO"+ 1SPUFJOUBSHFUJOHUPUIFCBDUFSJBMDZUPQMBTNJDNFNCSBOF Microbiol. Mol. Biol. Rev. 63 o #BVFS#8 4IFNFTI5 $IFO:3BQPQPSU5" "Ai1VTIBOE4MJEFw.FDIBOJTN "MMPXT4FRVFODF*OTFOTJUJWF5SBOTMPDBUJPOPG4FDSFUPSZ1SPUFJOTCZUIF4FD""51BTFCell 157 o 'FTTM5 8BULJOT% 0BUMFZ1 "MMFO8+ $PSFZ3" )PSOF+ #BMEXJO4" 3BEGPSE4& $PMMJOTPO *5VNB3 %ZOBNJDBDUJPOPGUIF4FDNBDIJOFSZEVSJOHJOJUJBUJPO QSPUFJOUSBOTMPDBUJPO BOEUFSNJOBUJPOElife 7. 67. 3.
(18) Chapter 3. 5TJSJHPUBLJ " $IBU[J ,& ,PVLBLJ . %F (FZUFS + 1PSUBMJPV "( 0SGBOPVEBLJ ( 4BSEJT .' 5SFMMF .# +SHFOTFO 5+% ,BSBNBOPV 4 &DPOPNPV " -POH-JWFE 'PMEJOH *OUFSNFEJBUFT1SFEPNJOBUFUIF5BSHFUJOH$PNQFUFOU4FDSFUPNFStructure 26 oF ,VTUFST*%SJFTTFO"+. 4FD" BSFNBSLBCMFOBOPNBDIJOFCell. Mol. Life Sci. 68, o. 68.
(19) 4FD:&(.FEJBUFT5SBOTMPDBUJPOPG4FDSFUPSZ1SPUFJO.PEJmFEXJUI/PO1PMZQFQUJEF4FHNFOUT. SUPPLEMENTARY INFORMATION. O N. Cl O. O EJPYP EJIZESP)QZSSPMZM QSPQBOPZMDIMPSJEF(1). "TPMVUJPOPG EJPYP EJIZESP)QZSSPMZM QSPQBOPJDBDJE NH NNPM JOUIJPOZMDIMPSJEF N- XBTSFnVYFEGPSI4VCTFRVFOUMZ UPMVFOF N- XBTBEEFE BOEUIFNJYUVSFXBTFWBQPSBUFEJOWBDVP5XPNPSFUPMVFOFFWBQPSBUJPODZDMFTXFSF QFSGPSNFEZJFMEJOHUIFQVSFQSPEVDU NH 1. )/.3 .)[ $%$M3 І E J )[ ) U J )[ ) U . 3.24 3.24 3.22 3.22 3.20. C (t) 3.22. 2.06. B (t) 3.82. 2.05. 3.84 3.82 3.82 3.81 3.80. A (d) 6.71. 2.00. 6.71 6.70. J )[ ) . 7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2 6.0 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 f1 (ppm). 69. 3.
(20) Chapter 3. O N. HN PEG3000 O. O Mal-PEG (2). To a solution of 1 (110 mg, 0,58mmol) in DCM (4 mL) was added alpha-Amino-omegaDBSCPYZ QPMZ FUIZMFOF HMZDPM IZESPDIMPSJEF 1&(.8 %BMUPO NH NNPM BOE%*1&" N- NNPM 5IFSFBDUJPONJYUVSFXBTTUJSSFEBU35VOEFS/2 atmosphere for 16h and subsequently all the volatiles were evaporated in vacuo5IF SFTJEVFXBTEJTTPMWFEJO.F0) N- BOEEJBMZTJT .8$0 JONFUIBOPMGPSI ZJFMEFEUIFQVSFQSPEVDU NH 1)/.3JOEJDBUFEUIBUUIFQFBLBUQQN indicative for compound 1 has been moved which is consistent with the formation of the EFTJSFEQSPEVDU<>. 7.4 7.2 7.0 6.8 6.6 6.4 6.2 6.0 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 f1 (ppm). 70.
(21) 4FD:&(.FEJBUFT5SBOTMPDBUJPOPG4FDSFUPSZ1SPUFJO.PEJmFEXJUI/PO1PMZQFQUJEF4FHNFOUT. REFERENCE +J 4 ;IV ; )PZF 53.BDPTLP $8.BMFJNJEF'VODUJPOBMJ[FE1PMZ Ї -caprolactone)block -poly(ethylene glycol) (PCL-PEG-MAL): Synthesis, Nanoparticle Formation, and Thiol $POKVHBUJPOMacromol. Chem. Phys. 210,o . 3. 71.
(22)
(23)
GERELATEERDE DOCUMENTEN
SUMMARY Cellular processes involve the activity of proteins as well as numerous interactions CFUXFFOQSPUFJOT*OCBDUFSJB
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.. Downloaded
University of Groningen The molecular choreography of the Sec translocation system Seinen, Anne-Bart
The research described in this thesis was carried out at the Groningen Biomolecular Sciences and Biotechnology (GBB) institute and Zernike Institute of Advanced Materials of
University of Groningen The molecular choreography of the Sec translocation system Seinen, Anne-Bart
At this point, signal recognition particle (SRP) and peptidyl- prolyl cis-trans isomerase trigger factor (TF) compete for binding the ribosome nascent chain 4,5. In
University of Groningen The molecular choreography of the Sec translocation system Seinen, Anne-Bart
To determine the cellular distribution of SecA, fluorescence signals from an image sequence spanning the first 7.5 seconds were averaged and a short-axis cross section was created
University of Groningen The molecular choreography of the Sec translocation system Seinen, Anne-Bart
Unlike SecA, SecE, SecF and YidC are integral membrane prorteins and thus their localization, under any set of conditions, will be restricted to the membrane, However, a
University of Groningen The molecular choreography of the Sec translocation system Seinen, Anne-Bart
To detect whether the diffusion of the translocon changes upon binding of nascent polypeptide chains and initiation of translocation, SecYEG-Atto 647N particles were imaged in
University of Groningen The molecular choreography of the Sec translocation system Seinen, Anne-Bart
There is only circumstantial evidence for the presence of such super complex in vivo, however, the single-molecule imaging method and analysis developed in chapter 2 allowed us
