University of Groningen Sec translocase in action Komarudin, Amalina Ghaisani

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(1)University of Groningen. Sec translocase in action Komarudin, Amalina Ghaisani. IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.. Document Version Publisher's PDF, also known as Version of record. Publication date: 2019 Link to publication in University of Groningen/UMCG research database. Citation for published version (APA): Komarudin, A. G. (2019). Sec translocase in action: Translocation initiation and processivity. University of Groningen.. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy 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 from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.. Download date: 28-06-2021.

(2) CHAPTER 2 (QJLQHHULQJRIWKH7ZRKHOL[ÀQJHURI SecA ATPase "NBMJOB(IBJTBOJ,PNBSVEJO *MKB,VTUFST 4BCSJOB,PDI "SOPME+.%SJFTTFO.

(3) Chapter 2. ABSTRACT 5IF4FDQBUIXBZJTUIFNBKPSSPVUFGPSQSPUFJOUSBOTQPSUBDSPTTBOEJOUPUIFDZUPQMBTNJD NFNCSBOFPGCBDUFSJB*OJUTNJOJNBMGPSN 4FDUSBOTMPDBTFDPOTJTUTPGUIF4FD:&( complex that functions as a membrane embedded protein conducting channel that associates with the peripheral membrane protein SecA which acts as an ATP-driven NPUPS *U IBT CFFO TVHHFTUFE UIBU UIF UXPIFMJY mOHFS )' PG 4FD" NBZ QVTI UIF QSFQSPUFJOUISPVHIUIFQPSFJOBiQPXFSTUSPLFiNBOOFS"MUFSOBUJWFMZ NPWFNFOUPGUIF )' NBZ ESJWF UIF PQFOJOH PG UIF 4FD:&( QPSF BOE USBOTMPDBUJPO NBZ PDDVS CZ B iNPMFDVMBSSBUDIFUiNFDIBOJTN)FSF XFFOHJOFFSFEUIF)'PG4FD"UPFYBNJOFUIF JNQPSUBODFPGUIF)'JOQSPUFJOUSBOTMPDBUJPO0VSEBUBEFNPOTUSBUFTGVODUJPOBMJUZPGB 4FD"WBSJBOUXJUIBTZNNFUSJDBMMZTIPSUFE)'CVUXJUIJODSFBTFEFOFSHFUJDSFRVJSFNFOUT. 40.

(4) &OHJOFFSJOHPGUIF5XPIFMJYmOHFSPG4FD""51BTF. INTRODUCTION Around one third of proteins produced in bacteria are either inserted or translocated BDSPTTUIFDZUPQMBTNJDNFNCSBOF5IFNBKPSTZTUFNGPSQSPUFJOUSBOTMPDBUJPOJTUIF4FD translocase which consists of the protein conducting channel SecYEG, the motor protein 4FD"BOEUIFBDDFTTPSZ4FD%'DPNQMFY<>5IFFOFSHZGPSUIFUSBOTMPDBUJPOQSPDFTT is provided by ATP hydrolysis via SecA, which drives stepwise transport of preproteins UISPVHI UIF USBOTMPDBUJPO DIBOOFM < > 5IF 4FD" "51BTF BDUJWJUZ JT BMMPTUFSJDBMMZ regulated by unfolded preproteins, the SecYEG complex, acidic phospholipids and SecB < >5IFDIBQFSPOF4FD#TUBCJMJ[FEGVMMZTZOUIFTJ[FEQSFQSPUFJOTJOBUSBOTMPDBUJPO DPNQFUFOUVOGPMEFETUBUFBOEUBSHFUFEUIFNUP4FD"CPVOEBUUIF4FD:&(DIBOOFM<> The nucleotide binding domains (NBDs) of SecA form a DEAD (Asp-Glu-Ala-Asp) motor, XIJDIJTBMTPGPVOEJO%/"3/"IFMJDBTFT< >#JOEJOHBOEIZESPMZTJTPG"51MFBEUP DPOGPSNBUJPOBMDIBOHFTOFDFTTBSZGPSQSFQSPUFJOUSBOTMPDBUJPO< >'VSUIFSNPSF 4FD" DPOUBJOTBQSFQSPUFJODSPTTMJOLJOHEPNBJOUIBUQMBZTSPMFJOTVCTUSBUFSFDPHOJUJPO<> UIFЃIFMJDBMTDBGGPMEEPNBJO UIFЃIFMJDBMXJOHEPNBJOBOEB$UFSNJOBMMJOLFS<> Structural studies on the Thermotoga maritima SecA-SecYEG complex [10] show that 4FD"QFOFUSBUFTUIF4FD:&(DIBOOFMWJBUIFTPDBMMFEUXPIFMJYmOHFS )' XIJDIBSF UXPTIPSUIFMJDFTUIBUBSFQBSUPGUIFЃIFMJDBMTDBGGPMEEPNBJO 'JH" 5IF)'PG4FD" is inserted into the cytoplasmic opening of the SecY channel, which brings the two helices JODMPTFQSPYJNJUZUPUIFUSBOTMPDBUFEQSFQSPUFJOBUUIFFOUSBODFPGUIF4FD:&(QPSF<> The mechanism of SecA mediated protein translocation through the SecYEG pore is MBSHFMZVOSFTPMWFE5XPNBJONFDIBOJTNTIBWFCFFOTVHHFTUFEUIFQPXFSTUSPLFBOE UIFNPMFDVMBSSBUDIFU5IFQPXFSTUSPLFNPEFMQSPQPTFTUIBUUIF)'BDUJWFMZQVTIFT UIFQSFQSPUFJOUISPVHI4FD:&(CZNFBOTPGIZESPQIPCJDJOUFSBDUJPOPGUIF)'XJUI UIFVOGPMEFEQPMZQFQUJEFDIBJO5IFMPPQPGUIF)'IBTBIJHIMZDPOTFSWFEUZSPTJOF SFTJEVF XIJDIJTDSVDJBMGPSUSBOTMPDBUJPOBOEDBOPOMZCFSFQMBDFECZCVMLZIZESPQIPCJD BNJOPBDJET<>*OUIFBOPUIFSNPEFM UIFNPMFDVMBSSBUDIFU UIF)'PG4FD"GVODUJPOT JOUIFPQFOJOHBOEDMPTVSFPGUIF4FD:&(DIBOOFM<>4JODFUIF)'JTJODPOUBDUXJUI the C4-loop between the transmembrane segments (TMS) 6 and 7 which are part of UIFTFDPOEIBMGPG4FD: NPWFNFOUTPGUIF)'DPVMEUSJHHFSUIFJOJUJBMPQFOJOHPGUIF 4FD:&(QPSF"MTP JOUIFBCTFODFPGBTUBCMF4FD"JOUFSBDUJPO GPSXBSEBOECBDLXBSE EJGGVTJPOPGQPMZQFQUJEFTUSFUDIFTXJUIJOUIF4FD:&(DIBOOFMNBZPDDVS< > )FSF XFUFTUFEUIFJNQPSUBODFPGUIF)'JOQSPUFJOUSBOTMPDBUJPOCZFYUFOEJOHBOE USVODBUJOHUIFMFOHUIPGUIF)'GPSVQUPBCPVUUXPIFMJDBMUVSOT0VSEBUBTIPXTUIBU. 41. 2.

(5) Chapter 2. 4FD"WBSJBOUXJUIBTZNNFUSJDBMMZTIPSUFE)'JTTUJMMGVODUJPOBMCVUSFRVJSFENPSFFOFSHZ GPSUSBOTMPDBUJPOQSPDFTT. Fig. 1. Overview of SecA 2HF variants. (A) A representation of the T. maritima SecA-SecYEG viewed from the side [10]; SecYEG (purple), SecA : NBD1 (blue), NBD2 (light blue), PPXD (green), )8% PSBOHF )4% QJOL )' SFE 4FD"QSPUPNFSTIPXOJOHSBZ # 5IFTUSVDUVSFPG)' SecA with insertion region (yellow) and the conserved Tyr794 (magenta), Alanine residue shown JODZBO $ 4DIFNBUJDSFQSFTFOUBUJPOBOEDPOTUSVDUJPOPGFYUFOEFEBOEUSVODBUFE)'4FD" %FMFUFEBNJOPBDJETBSFTIPXOBTEBTIFEMJOFT $ (SPXUIDPNQMFNFOUBUJPOPGE.coli#- XJUIQMBTNJEFODPEFE4FD")'NVUBOUT$FMMTXFSFTQPUUFEPOUXP-#BNQJDJMMJOBHBSQMBUFT XIJDIXFSFJODVCBUFEBUQFSNJTTJWFBOEOPOQFSNJTTJWFUFNQFSBUVSF%JMVUJPOGBDUPSTBSFJOEJDBUFE CFMPXUIFQBOFMT. MATERIAL AND METHODS Plasmids construction The secAHFOFXBTBNQMJmFEGSPNE. coli ,HFOPNJD%/"CZ1$3BOEUIFGSBHNFOU was digested with NdeI and Xho*BOEDMPOFEJOUPEJHFTUFEQ&5CUPDSFBUFQ"(, Unique restriction site MunI and BseRI in pAGK002 were then used to insert synthetic EPVCMFTUSBOEFE%/"GSBHNFOUT *OWJUSPHFO DPOUBJOJOHUIFEFTJSFETFRVFODFTnBOLFE. 42.

(6) &OHJOFFSJOHPGUIF5XPIFMJYmOHFSPG4FD""51BTF. by these restriction sites, resulting in pAGK003, pAGK004, pAGK005, pAGK006, Q"(, Q"(, Q"(, BOE Q"(, "MM DPOTUSVDUT XFSF DPOmSNFE CZ TFRVFODJOH 2YHUH[SUHVVLRQVDQGSXULÀFDWLRQVRI6HF$PXWDQWV *OOFSNFNCSBOFWFTJDMFT *.7T DPOUBJOJOHPWFSFYQSFTTFE4FD:&(XFSFJTPMBUFEBT EFTDSJCFE<>8JMEUZQF4FD" BOE4FD"åXFSFPWFSFYQSFTTFEJOE. coli BL21 (pLySs) at 37 ºC, but SecA i3 mutant was expressed at 25 ºC to prevent the forming of inclusion CPEJFT4FD" 4FD#BOEQSP0NQ"XFSFQVSJmFEBTEFTDSJCFE<>QSP0NQ" $4 XBTMBCFMMFEXJUI'MVPSFTDFJONBMFJNJEF .PMFDVMBS1SPCFT &VHFOF 03 64" <> In vivo complementation assay E. coli#-<>XBTVTFEUPUFTUUIFJOWJWPGVODUJPOBMJUZPG4FD"NVUBOU<>#SJFnZ QMBTNJETFODPEJOHGPSXJMEUZQF4FD" 4FD")'NVUBOUTBOEFNQUZWFDUPSQ&5C XFSFUSBOTGPSNFEJOUPUIF4FD"UTNVUBOUTUSBJO$FMMTXFSFHSPXOPWFSOJHIUJO-#XJUI HMVDPTF XW BOEBNQJDJMMJO HN- BU¡$5IFDVMUVSFTXFSFEJMVUFEJO-# XJUIHMVDPTF XW BOEBNQJDJMMJO 0%ON BOEJODVCBUFEBU¡$GPS IPVST*UGVSUIFSDPOUJOVFEXJUITQPUUJOHçMPGBGPMETFSJBMEJMVUJPOPGDVMUVSFTPOUXP -#BNQJDJMMJOBHBSQMBUFTBOEJODVCBUFEBUUIFQFSNJTTJWFUFNQFSBUVSFPG¡$PSBUUIF OPOQFSNJTTJWFUFNQFSBUVSFPG¡$ In vitro translocation assay 5SBOTMPDBUJPO"51BTFBDUJWJUZJOVSFBXBTIFE*.7TPGXJMEUZQF4FD"BOE4FD"NVUBOUT were determined by measuring the amount of released free phosphate using the malachite green assay [17]. In vitroUSBOTMPDBUJPOPGQSP0NQ"nVPSFTDFJOMBCFMMFEXFSF DBSSJFEPVUBTQSFWJPVTMZ<>XJUI*.7TXBTIFEXJUI.VSFB GPMMPXFECZ4%4o1"(& nVPSFTDFODFJNBHJOHBOERVBOUJmFEVTJOH*NBHF+TPGUXBSF SecA binding assay Microscale Thermophoresis - The dissociation constants (KD) of wild type SecA, SecA i3 BOE4FD"åXFSFNFBTVSFEVTJOHUIF.POPMJUI/5GSPN/BOPUFNQFS5FDIOPMPHJFT BTEFTDSJCFE IPXFWFS MJQJETXFSFTPOJDBUFEBOETPMVCJMJ[FEVTJOH5SJUPOJOTUFBE PG/BDIPMBUF<> ANS binding to SecA - ANS binding assay was performed to gain more insight into the HMPCBMGPMEJOHTUBUFPG4FD")'NVUBOUT4FD"UP"/4NPMBSSBUJPPGXBTVTFE BOEUIF"/4nVPSFTDFODFJOUFOTJUZXBTNFBTVSFEBU$VTJOH2VBOUB.BTUFS. 43. 2.

(7) Chapter 2. TQFDUSPnVPSJNFUFS 1IPUPO5FDIOPMPHZ*OUFSOBUJPOBM GSPNONUPONXJUIBO FYDJUBUJPOXBWFMFOHUIPGON. RESULTS Truncation and extension of the 2HF of SecA 5PFYBNJOFIPXUIF)'PG4FD"JTJOWPMWFEJOQSPUFJOUSBOTMPDBUJPO BTFUPG4FD" NVUBOUTXJUIFYUFOEFEBOEUSVODBUFE)'XFSFDSFBUFE5IF)'DPOTJTUTPGUXPTIPSU helices that are connected through a loop which harbors the highly conserved tyrosine SFTJEVF 'JH# "OBMJHONFOUBOBMZTJTXBTQFSGPSNFEUPJEFOUJGZSFHJPOTJOUIF)' for the parallel insertion of an equal number of alanine residues into the two helices and MJLFXJTF UIFEFMFUJPOPGBOFRVBMOVNCFSPGSFTJEVFTJOPSEFSUPTIPSUFOUIFUXPIFMJDFT The alignment showed that D772-S773 and E816-S817 are not conserved thus this region NPSFMJLFMZUPBDDFQUNVUBUJPOT5IF)'XBTNPEJmFECZJOUSPEVDJOHVQUPTJYBMBOJOF SFTJEVFTJOFBDIIFMJY SFTVMUJOHJOBOFYUFOTJPOPGVQUPUXPIFMJDBMUVSOT'PSUSVODBUJPO PGIFMJDFTJOUIF)'NPUJG BNJOPBDJETBSPVOEUIFMFTTDPOTFSWFESFHJPOXFSFEFMFUFE VQUPUXPIFMJDBMUVSOT 'JH$ "MMNVUBOUTBSFMJTUFEJO5BCMF Table 1 | Strains and plasmids used in this study Strains/plasmids. Description. Source. E. coli%)Ѓ. TVQ& åMBD6 åMBD;@. ITE3 SFD" FOE" HZS" UIJSFM". [19]. E. coli BL21(DE3) pLysS. F-ompT hsdSB(rB-, mB-) gal dcm Ѝ%& Q-ZT4 $BN3. [20]. E. coli#-. [secA13(Am) supF(Ts) trp(Am) zch::Tn10 recA::CAT clpA,"/ Ѝ%& >. [15]. E. coli SF100. ' åMBD9 HBM& HBM, UIJ SQT- TUS" åQIP" QWV** åPNQ5. [21]. pAGK002. 8JMEUZQF4FD"JOQ&5C. This study. pAGK003. )'NVUBOU4FD"XJUI"MBOJOFJOTFSUJPOCFUXFFO% and S773 and E816 and S817 in pET20b (i1). This study. pAGK004. )'NVUBOU4FD"XJUI"MBOJOFJOTFSUJPOCFUXFFO% and S773 and E816 and S817 in pET20b (i2). This study. pAGK005. )'NVUBOU4FD"XJUI"MBOJOFJOTFSUJPOCFUXFFO% and S773 and E816 and S817 in pET20b (i3). This study. pAGK006. )'NVUBOU4FD"XJUI"MBOJOFJOTFSUJPOCFUXFFO% and S773 and E816 and S817 in pET20b (i6). pAGK007. )'NVUBOU4FD"XJUIBEFMFUJPOPG4BOE4JO Q&5C å. pAGK008. )'NVUBOU4FD"XJUIEFMFUJPOTPG4 -BOE4 This study -JOQ&5C å. 44. This study.

(8) &OHJOFFSJOHPGUIF5XPIFMJYmOHFSPG4FD""51BTF. Table 1 | Continued pAGK009. )'NVUBOU4FD"XJUIEFMFUJPOTPG%-BOE &-JOQ&5C å. This study. pAGK010. )'NVUBOU4FD"XJUIEFMFUJPOTPG58BOE .,JOQ&5C å. This study. 5IFGVODUJPOBMJUZPGUIF)'NVUBOUTPG4FD"XBTUFTUFEin vivo by a complementation BTTBZ"UFNQFSBUVSFTFOTJUJWF4FD"NVUBOUE. coli #-XBTVTFEBTBIPTUGPSUIF QMBTNJETDPOUBJOJOHUIFHFOFTGPSUIF4FD"WBSJBOUTPSXJMEUZQF4FD"*OUIJT4FD"UT strain, chromosomal secA expression can be shut off by growth at 42 °C because of the presence of a secA BNCFSNVUBUJPOBOEBUFNQFSBUVSFTFOTJUJWFBNCFSTVQQSFTTPS<> 5IF4FD"EFmDJFODZDBOCFDPNQMFNFOUFECZUIFFYQSFTTJPOPGBGVODUJPOBMQMBTNJE CPSOF4FD""MMNVUBOUTPG4FD"TIPXFEOPSNBMHSPXUIBUUIFQFSNJTTJWFUFNQFSBUVSF $ "UOPOQFSNJTTJWFUFNQFSBUVSF $ XJMEUZQF4FD" 4FD"åBOE4FD"å XFSFBCMFUPDPNQMFNFOUUIFHSPXUIEFGFDUPGUIF4FD"UTTUSBJO 'JH% 5IJTSFTVMU TVHHFTUTUIBU4FD"åBOE4FD"åBSFGVODUJPOBMXIJMFUIFPUIFS4FD")'WBSJBOUTBSF EFGFDUJWF'PSGVSUIFSDIBSBDUFSJ[BUJPOPGUIF4FD"WBSJBOUTin vitro, we focused hereafter PO4FD"JBOE4FD"åBTUIFSFQSFTFOUBUJWFNVUBOUT Truncation of 2HF by about one helical turn does not interfere with translocation The ATPase activity of SecA is strongly stimulated when both SecYEG and preproteins are QSFTFOU BOBDUJWJUZUFSNFE4FD"USBOTMPDBUJPO"51BTF<>*OPSEFSUPUFTUUIF"51BTF BDUJWJUZPGUIF4FD")'NVUBOUTin vitro,XFQVSJmFEUIFXJMEUZQF4FD" 4FD"JBOE 4FD"åGSPNPWFSFYQSFTTJOHE. coliTUSBJOT 'JH"# "EEJUJPOPGUIFQSFQSPUFJO QSP0NQ"UPBSFBDUJPOPG4FD"XJUIVSFBXBTIFE*.7TPGE. coli resulted in strong TUJNVMBUJPOPGUIF"51BTFBDUJWJUZPGCPUIUIFXJMEUZQF4FD"BOE4FD"å*ODPOUSBTU UIF"51BTFTUJNVMBUJPOPG4FD"JXBTMPX BMCFJUEFUFDUBCMFBCPWFCBDLHSPVOE 'JH$. 45. 2.

(9) Chapter 2. Fig. 2. The effect of extension and truncation in 2HF SecA in protein translocation. (A) 0WFSFYQSFTTJPOPGXJMEUZQF4FD" -BOF 4FD"J -BOF BOE4FD"å -BOF JO&DPMJ #- Ѝ%& Q-ZT4"TUIFDPOUSPM *15(XBTPNJUUFEJOMBOF BOE # $PPNBTTJFTUBJOFE 4%41"(&PGQVSJmFEXJMEUZQF4FD" -BOF 4FD"J -BOF BOE4FD"å -BOF .PMFDVMBS NBTTFTPGQSPUFJOTUBOEBSEBSFJOEJDBUFEPOUIFMFGU $ 4FD""51BTFBDUJWJUZJOVSFBXBTIFE *.7TJOUIFBCTFODF XIJUFCBST BOEUIFQSFTFODF CMBDLCBST PGQSP0NQ" % 5SBOTMPDBUJPOPG nVPSFTDFJOMBCFMMFEQSP0NQ"JOUPVSFBXBTIFE*.7TXJUI8JMEUZQF4FD"BOE4FD")'NVUBOUT 5IFTPMJEMJOFTQSFTFOUB.JDIBFMJT.FOUFOmU. 'VSUIFS USBOTMPDBUJPO PG QSP0NQ" JOUP UIF VSFB TUSJQQFE *.7T XBT BTTBZFE CZ JUT QSPUFDUJPO BHBJOTU QSPUFJOBTF , 5IFTF *.7T BSF EFGFDUJWF JO QSPUPO NPUJWF GPSDF generation through ATP hydrolysis and thus translocation is solely coupled to the "51BTFBDUJWJUZPG4FD"4FD"JDPVMEPOMZUSBOTMPDBUFNJOPSBNPVOUTPGQSP0NQ" XIJMF4FD"åTIPXFEBSFEVDFEUSBOTMPDBUJPOBDUJWJUZDPNQBSFEUPUIFXJMEUZQF4FD" CVUBTJNJMBS4FD"EFQFOEFODF 'JH% 5IFTFSFTVMUTBSFJOHPPEBHSFFNFOUXJUIUIF in vivo complementation data and demonstrate the importance and promiscuity of the MFOHUIPG)'. 46.

(10) &OHJOFFSJOHPGUIF5XPIFMJYmOHFSPG4FD""51BTF. 6HF$¨ELQGVWR6HF<(*ZLWKKLJKDIÀQLW\ 5P HBJO NPSF JOTJHIU JOUP UIF JOUFSBDUJPO CFUXFFO 4FD" )' NVUBOUT BOE 4FD:&( XF FYBNJOFE UIF CJOEJOH VTJOH .JDSPTDBMF 5IFSNPQIPSFTJT .45 5IJT UFDIOJRVF is applicable to analyze protein-protein interactions based on their movement in a UFNQFSBUVSFHSBEJFOU5IFNPUJPOPGUIFVOCPVOENPMFDVMFTJOUIFUFNQFSBUVSFHSBEJFOU is different than the bound molecules due to their distinct charge, hydration shell and NPMFDVMBSTJ[F< >5IVT UIFCPVOETUBUFTBOEUIFVOCPVOETUBUFTDBOCFTFQBSBUFE and therefore the dissociation constant K D PCUBJOFE *O PSEFS UP EP NPUJPO CBTFE FYQFSJNFOUTXFSFDPOTUJUVUFE4FD:&(JOUPIPNPHFOPVTMZTJ[FEMJQJEOBOPEJTDT 0VSQSFWJPVTXPSLTIPXFEUIBU4FD"CJOETWJBJUT/UFSNJOVTUPBOJPOJDMJQJET XIJDI QSJNFT 4FD" GPS IJHI BGmOJUZ CJOEJOH UP 4FD:&( <> 5P FOTVSF B IJHI MJQJE DPOUFOU surrounding SecYEG and enough space for the SecA N-terminus to bind, large nanodiscs CFBSJOHBTJOHMF4FD:&(DPNQMFYXFSFVTFEJOUIJTTUVEZUPNPOJUPS4FD"CJOEJOH 5IFnVPSFTDFODFJOUFOTJUZPG4FD:&(MBSHFOBOPEJTDTBUBDPOTUBOUDPODFOUSBUJPOXBT PCTFSWFEXIJMFVOMBCFMFE4FD"XBTUJUSBUFEUPBmOBMDPODFOUSBUJPOPG."TTIPXO JO'JH" XJMEUZQF4FD"BOE4FD"åCJOEUP4FD:&(MBSHFEJTDTXJUIBOBQQBSFOU KDPGO.BOEO. SFTQFDUJWFMZ XIJMF4FD"JTIPXFEOP JOUFSBDUJPO. Fig. 3. Analysis the binding of SecA 2HF mutants (A) MST measurements of wild type SecA CMBDL 4FD"J XIJUF BOE4FD"å HSFZ 5IFDVSWFTXFSFmUUFEVTJOHUIF)JMMFRVBUJPOBOE apparent KDWBMVFTXFSFEFUFSNJOFE&SSPSCBSTSFQSFTFOUUIFTUBOEBSEFSSPSPGNFBTVSFNFOUT # "/44FD"CJOEJOHBTTBZ8JMEUZQF4FD" CMBDLTPMJEMJOFT 4FD"J CMBDLEBTIFTMJOFT 4FD" å CMBDLEPUUFEMJOFT BOEPOMZ"/4BTDPOUSPM HSFZTPMJEMJOFT . 5IFBCPWFSFTVMUTEFNPOTUSBUFUIBU4FD"JJTEFGFDUJWFJOJUTGVODUJPO5PWBMJEBUFQSPQFS GPMEJOHPGUIF4FD")'NVUBOUT "/4CJOEJOHBTTBZTXFSFQFSGPSNFE8IFO"/4CJOET to hydrophobic residues of protein, the emission maximum of ANS will be shifted to blue. 47. 2.

(11) Chapter 2. XBWFMFOHUISBOHF<>8JMEUZQF4FD"BOE4FD"åTIPXFETJNJMBS"/4nVPSFTDFODF TQFDUSB CVU4FD"JTIPXTBCMVFTIJGU'JH#5IJTJOEJDBUFTUIBU4FD"JIBTNPSF IZESPQIPCJDTVSGBDFFYQPTFEUPUIFTPMVUJPOBOEUIVTJTNJTGPMEFE. DISCUSSION The mechanism of SecA mediated protein translocation through the SecYEG pore is TUJMMQPPSMZVOEFSTUPPE'SPNUIFDPTUSVDUVSFPGT.maritima SecA–SecYEG [10], at least UXPQPTTJCMFNFDIBOJTNTGPSQSPUFJOUSBOTMPDBUJPODBOCFEFSJWFE*OUIFDPTUSVDUVSF UIF)'PG4FD"JTJOTFSUFEJOUPUIFDZUPQMBTNJDPQFOJOHPGUIF4FD:DIBOOFM BOEJU IBTCFFOTVHHFTUFEUIBUJUNBZQVTIUIFQSFQSPUFJOUISPVHIUIF QPSFMJLFBiQPXFS TUSPLFi <> BT UIF )' DBO CF DSPTTMJOLFE UP USBOTMPDBUJOH QSFQSPUFJOT 5IF PUIFS NPEFM UIFiNPMFDVMBSSBUDIFUw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åBOE4FD" åBSFBCMFUPDPNQMFNFOU4FD"UTTUSBJO TVHHFTUJOHUIFnFYJCJMJUZPG)'UPQSFTFSWF UIFIFMJDBMTUSVDUVSF4FD"åBMTPTIPXFEUSBOTMPDBUJPOPGQSP0NQ"in vitro0VSEBUB DMFBSMZEFNPOTUSBUFUIBUUIFTIPSUFOJOHPGUIF)'CZBCPVUPOFIFMJDBMUVSOSFTVMUTJOB GVODUJPOBM4FD"WBSJBOU 'VSUIFS UIJTTUVEZBMTPQSPWJEFTJOGPSNBUJPOBCPVUUIFJOUFSBDUJPOTPG4FD")'NVUBOUT BOEUIF4FD:&(DIBOOFM The lipid environment around SecYEG and the N-terminus of 4FD"BSFJNQPSUBOUGBDUPSTGPSUIFIJHIBGmOJUZCJOEJOHPG4FD"UP4FD:&(< >5IVT XFTQFDVMBUFUIBUUIFFYUFOTJPOBOEUSVODBUJPOPGUIF)'TIPVMEOPUBGGFDUUIFCJOEJOH PG4FD"4FD:&(*OEFFE 4FD"åJOUFSBDUTXJUI4FD:&(JOEJTUJOHVJTIBCMFGSPNXJME UZQF4FD"5IFMPOHFSMPPQPGUIF)'PG4FD"J IPXFWFS JOUFSGFSFTXJUIUIFEPDLJOH UP4FD:&(.PSFPWFS UIFCMVFTIJGUPG"/4CPVOE4FD"JNBZBMTPJOEJDBUFNJTGPMEJOH PGUIJTNVUBOU5IJTTVHHFTUTUIBUBQSPQFSGPMEJOHPGUIF)'JTDSJUJDBMUPUIFPWFSBMM GPMEJOHPGUIF4FD"QSPUFJO 8F PCTFSWFE UIBU UIF USBOTMPDBUJPO "51BTF PG 4FD"å JT OFBS UP FRVBM PS TMJHIUMZ enhanced as compared to that of wild type SecA, whereas the translocation activity. 48.

(12) &OHJOFFSJOHPGUIF5XPIFMJYmOHFSPG4FD""51BTF. JT TMJHIUMZ SFEVDFE *O B NPEFM XIFSF UIF )' PG 4FD" GVODUJPOT BT B MFWFS UP QVTI QSPUFJOTUISPVHI4FD:&( UIFSFBDIPGBTIPSUFOFE)'BTXJUI4FD"å BTNBMMFS TUFQQJOHTJ[FXPVMECFFYQFDUFE5IJTXPVMEMFBEUPBOFOIBODFE"51SFRVJSFNFOU per translocation event as compared to the wild type SecA [28], as observed with the 4FD"åWBSJBOU)PXFWFS UIFGVODUJPOPGUIF)'BTBUSBOTMPDBUJPOMFWFSJTDIBMMFOHFE CZ UIF PCTFSWBUJPO UIBU JNNPCJMJ[BUJPO PG )' XJUI B MPPQ JO 4FD: EPFT OPU QFSUVSC USBOTMPDBUJPO<>'VSUIFSNPSF USBOTMPDBUJPONBZCFBDPNCJOBUJPOPGCPUINFDIBOJTNT JF UIFQPXFSTUSPLFBOENPMFDVMBSSBUDIFUBTTMJEJOHPGUIFQSFQSPUFJOUISPVHIUIFQPSF IBTCFFOPCTFSWFEJOUIFBCTFODFPGB4FD"JOUFSBDUJPO< >'PSJOTUBODF UIF)' of SecA may interact with the polypeptide ensuring a one directional movement through UIF4FD:&(QPSFWJBEJGGVTJPOSBUIFSUIBOBDUJOHBTBMFWFS. ACKNOWLEDGEMENTS 8FXPVMEMJLFUPUIBOL*1SBCVEJBOTZBIBOE"#4FJOFOGPSUIFUFDIOJDBMBTTJTUBODF 5IJTXPSLXBTGVOEFECZ5IF/FUIFSMBOET0SHBOJ[BUJPOGPS4DJFOUJmD3FTFBSDI /80 . 49. 2.

(13) Chapter 2. REFERENCES %SJFTTFO"+./PVXFO/ 1SPUFJO5SBOTMPDBUJPO"DSPTTUIF#BDUFSJBM$ZUPQMBTNJD .FNCSBOFAnnu. Rev. Biochem. 77 o 4DIJFCFM& %SJFTTFO"+ )BSUM'68JDLOFS8 %FMUBNV)

(14) BOE"51GVODUJPOBU EJGGFSFOUTUFQTPGUIFDBUBMZUJDDZDMFPGQSFQSPUFJOUSBOTMPDBTFCell 64 o WBOEFS8PML+ ,MPTF. #SFVLJOL& %FNFM3" EF,SVJKGG# 'SFVEM3%SJFTTFO"+ Characterization of a Bacillus subtilis4FD"NVUBOUQSPUFJOEFmDJFOUJOUSBOTMPDBUJPO"51BTF BOESFMFBTFGSPNUIFNFNCSBOFMol. Microbiol. 8 o 1BQBOJLPV& ,BSBNBOPV4&DPOPNPV" #BDUFSJBMQSPUFJOTFDSFUJPOUISPVHIUIF USBOTMPDBTFOBOPNBDIJOFNat. Rev. Microbiol. 5 o ,PDI4 EF8JU+( 7PT* #JSLOFS+1 (PSEJJDIVL1 )FSSNBOO" WBO0JKFO".%SJFTTFO "+. -JQJET"DUJWBUF4FD"GPS)JHI"GmOJUZ#JOEJOHUPUIF4FD:&($PNQMFYJ. Biol. Chem. 291 o 'FLLFT1 EF8JU+( WBOEFS8PML+1 ,JNTFZ)) ,VNBNPUP$"%SJFTTFO"+ Preprotein transfer to the Escherichia coli translocase requires the co-operative binding of 4FD#BOEUIFTJHOBMTFRVFODFUP4FD"Mol. Microbiol. 29 o )VOU +' 8FJOLBVG 4 )FOSZ - 'BL ++ .D/JDIPMBT 1 0MJWFS %# %FJTFOIPGFS + /VDMFPUJEFDPOUSPMPGJOUFSEPNBJOJOUFSBDUJPOTJOUIFDPOGPSNBUJPOBMSFBDUJPODZDMFPG4FD" Science 297 o 4BUP, .PSJ) :PTIJEB..J[VTIJNB4 $IBSBDUFSJ[BUJPOPGBQPUFOUJBMDBUBMZUJD SFTJEVF "TQ JOUIFIJHIBGmOJUZ"51CJOEJOHTJUFPGEscherichia coli SecA, translocation "51BTFJ. Biol. Chem. 271 o #BVFS#83BQPQPSU5" .BQQJOHQPMZQFQUJEFJOUFSBDUJPOTPGUIF4FD""51BTFEVSJOH USBOTMPDBUJPOProc. Natl. Acad. Sci. U. S. A. 106 o ;JNNFS+ /BN:3BQPQPSU5" 4USVDUVSFPGBDPNQMFYPGUIF"51BTF4FD"BOEUIF QSPUFJOUSBOTMPDBUJPODIBOOFMNature 455 o &SMBOETPO,+ .JMMFS4#. /BN: 0TCPSOF"3 ;JNNFS+3BQPQPSU5" "SPMFGPSUIF UXPIFMJYmOHFSPGUIF4FD""51BTFJOQSPUFJOUSBOTMPDBUJPONature 455 o "MMFO8+ $PSFZ3" 0BUMFZ1 4FTTJPOT3# 3BEGPSE4& 5VNB3$PMMJOTPO* 5XP way communication between SecY and SecA suggests a Brownian ratchet mechanism for QSPUFJOUSBOTMPDBUJPOElife 5 ,VTUFST* WBOEFO#PHBBSU( EF8JU+ ,SBTOJLPW7 1PPMNBO#%SJFTTFO" 1VSJmDBUJPO BOEGVODUJPOBMSFDPOTUJUVUJPOPGUIFCBDUFSJBMQSPUFJOUSBOTMPDBUJPOQPSF UIF4FD:&(DPNQMFY Methods Mol. Biol. 619 o ,FZ[FS+%F %PFT$7BO%FS%SJFTTFO"+. ,JOFUJD"OBMZTJTPGUIF5SBOTMPDBUJPOPG Fluorescent Precursor Proteins into Escherichia coli.FNCSBOF7FTJDMFT 277 o .JUDIFMM$0MJWFS% 5XPEJTUJODU"51CJOEJOHEPNBJOTBSFOFFEFEUPQSPNPUFQSPUFJO export by Escherichia coli4FD""51BTFMol. Microbiol. 10 o /B# :PV; :BOH)5BJ1$ $IBSBDUFSJ[BUJPOPGUIFNJOJNBMMFOHUIPGGVODUJPOBM4FD" in Escherichia coliBiochem. Biophys. Res. Commun. 456 o. 50.

(15) &OHJOFFSJOHPGUIF5XPIFMJYmOHFSPG4FD""51BTF -JMM 3 $VOOJOHIBN , #SVOEBHF -" *UP , 0MJWFS % 8JDLOFS 8 4FD" QSPUFJO hydrolyzes ATP and is an essential component of the protein translocation ATPase of Escherichia coliEMBO J. 8 o 7BOEFS%PFT$ %F,FZ[FS+ 7BOEFS-BBO.%SJFTTFO"+. 3FDPOTUJUVUJPOPG 1VSJmFE#BDUFSJBM1SFQSPUFJO5SBOTMPDBTFJO-JQPTPNFTMethods Enzymol. 372 o )BOBIBO% 4UVEJFTPOUSBOTGPSNBUJPOPGEscherichia coliXJUIQMBTNJETJ. Mol. Biol. 166 o ;IBOH94UVEJFS'8 .FDIBOJTNPGJOIJCJUJPOPGCBDUFSJPQIBHF53/"QPMZNFSBTF CZ5MZTP[ZNFJ. Mol. Biol. 269 o /PVXFO / WBO EFS -BBO . %SJFTTFO "+ 4FD%'ZBK$ JT OPU SFRVJSFE GPS UIF NBJOUFOBODFPGUIFQSPUPONPUJWFGPSDFFEBS Lett. 508 o +JMBWFBOV-0MJWFS% In vivo membrane topology of Escherichia coli SecA ATPase reveals extensive periplasmic exposure of multiple functionally important domains clustering POPOFGBDFPG4FD"J. Biol. Chem. 282 o 8JFOLFO$+ #BBTLF1 3PUICBVFS6 #SBVO%%VIS4 1SPUFJOCJOEJOHBTTBZTJO CJPMPHJDBMMJRVJETVTJOHNJDSPTDBMFUIFSNPQIPSFTJTNat. Commun. 1 4FJEFM4"* %JKLNBO1. -FB8" WBOEFO#PHBBSU( +FSBCFL8JMMFNTFO. -B[JD" +PTFQI +4 4SJOJWBTBO1 #BBTLF1 4JNFPOPW" ,BUSJUDI* .FMP'" -BECVSZ+& 4DISFJCFS( 8BUUT " #SBVO%%VIS4 .JDSPTDBMFUIFSNPQIPSFTJTRVBOUJmFTCJPNPMFDVMBSJOUFSBDUJPOT VOEFSQSFWJPVTMZDIBMMFOHJOHDPOEJUJPOTMethods 59 o )BXF " 4VUUFS . +JTLPPU 8 &YUSJOTJD nVPSFTDFOU EZFT BT UPPMT GPS QSPUFJO DIBSBDUFSJ[BUJPOPharm. Res. 25 o ,VTUFST*%SJFTTFO"+. 4FD" BSFNBSLBCMFOBOPNBDIJOFCell. Mol. Life Sci. 68, o )FOESJDL+18JDLOFS8 4FD"QSPUFJOOFFETCPUIBDJEJDQIPTQIPMJQJETBOE4FD:& QSPUFJOGPSGVODUJPOBMIJHIBGmOJUZCJOEJOHUPUIFEscherichia coliQMBTNBNFNCSBOFJ. Biol. Chem. 266 o /PVXFO/ #FSSFMLBNQ(%SJFTTFO"+. $IBSHFEBNJOPBDJETJOBQSFQSPUFJOJOIJCJU 4FD"EFQFOEFOUQSPUFJOUSBOTMPDBUJPOJ. Mol. Biol. 386 o 8IJUFIPVTF4 (PME7B. 3PCTPO" "MMFO8+ 4FTTJPOT3#$PMMJOTPO* .PCJMJUZPG UIF4FD"IFMJYmOHFSJTOPUFTTFOUJBMGPSQPMZQFQUJEFUSBOTMPDBUJPOWJBUIF4FD:&(DPNQMFY J. Cell Biol. 199 o #BVFS#8 4IFNFTI5 $IFO:3BQPQPSU5" "Ai1VTIBOE4MJEFw.FDIBOJTN "MMPXT4FRVFODF*OTFOTJUJWF5SBOTMPDBUJPOPG4FDSFUPSZ1SPUFJOTCZUIF4FD""51BTFCell 157 o. 51. 2.

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