University of Groningen Sec translocase in action Komarudin, Amalina Ghaisani
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(2) CHAPTER 5 Summary Samenvatting Ringkasan.
(3) Chapter 5. 94.
(4) Summary. SUMMARY Cellular processes involve the activity of proteins as well as numerous interactions CFUXFFOQSPUFJOT*OCBDUFSJB QSPUFJOTBSFTZOUIFTJ[FEBUSJCPTPNFTJOUIFDZUPTPM" TVCTUBOUJBMTIBSFPGUIFQSPUFJOT IPXFWFS GVODUJPOPVUTJEFUIFDZUPTPM'PSJOTUBODF JO Escherichia coli, they either need to be translocated across the cytoplasmic membrane to reach the periplasm or outer membrane, or they need to be integrated into the DZUPQMBTNJDNFNCSBOF5IF4FDQBUIXBZJTUIFNBKPSSPVUFGPSQSPUFJOUSBOTQPSUBDSPTT BOEJOUPUIFDZUPQMBTNJDNFNCSBOF< >5IJTQBUIXBZJTGPSNFECZUIF4FDUSBOTMPDBTF which consists of a protein-conducting channel in the cytoplasmic membrane formed by the SecYEG heterotrimeric membrane protein complex [3], the motor protein SecA which utilizes ATP to drive the translocation process [4–6], and the accessory heterotrimeric NFNCSBOFQSPUFJODPNQMFY4FD%'ZBK$UIBUDPVQMFTUSBOTMPDBUJPOUPUIFQSPUPONPUJWF GPSDF<> 5PMPDBMJ[FQSPUFJOTUPUIF4FDUSBOTMPDBTF UXPEJGGFSFOUQBUIXBZTFYJTU4FDSFUPSZQSPUFJO are synthesized with an N-terminal signal sequence that directs them in an unfolded state to SecYEG-bound SecA protein, either with or without the help of the molecular chaperone 4FD#< >5IJTSPVUFJTBMTPUFSNFEUIFQPTUUSBOTMBUJPOBMQBUIXBZBTUIFUSBOTMPDBUJPOPG UIFTFQSPUFJOTJTOPUDPVQMFEUPDIBJOFMPOHBUJPOBUUIFSJCPTPNF$ZUPQMBTNJDNFNCSBOF QSPUFJOTBSFSFDPHOJ[FECZUIFJSmSTUIZESPQIPCJDUSBOTNFNCSBOFTFHNFOUEVSJOHUIFJS USBOTMBUJPOCZUIFSJCPTPNF XIFSFUIFZBSFCPVOECZTJHOBMSFDPHOJUJPOQBSUJDMF 431 5IJTUFSOBSZDPNQMFYPGSJCPTPNF 431BOEOBTDFOUNFNCSBOFQSPUFJOJTTVCTFRVFOUMZ DPUSBOTMBUJPOBMMZUBSHFUFEUPUIFNFNCSBOFUPCJOEUPUIF431SFDFQUPS 'UT:<>/FYU (51CJOEJOHBOEIZESPMZTJTCZUIFIFUFSPEJNFSJD431'UT:DPNQMFYSFTVMUTJOBSFMFBTF of the nascent membrane protein to the SecYEG complex for membrane integration, a QSPDFTTUIBUJTDPVQMFEUPDIBJOFMPOHBUJPOBUUIFSJCPTPNF:JE$NBZBTTJTU4FD:&(JO NFNCSBOFQSPUFJOJOTFSUJPO<>#PUIQBUIXBZTIBWFCFFOFYUFOTJWFMZTUVEJFEJOMBTUGFX EFDBEFTCZBDPNCJOBUJPOPGHFOFUJD CJPDIFNJDBMBOETUSVDUVSBMNFUIPET0VSDVSSFOU understand of the functional and structural characteristics of the SecYEG and SecA are summarized in chapter 1PGUIFUIFTJT " NBKPS BEWBODF JO PVS VOEFSTUBOEJOH PG UIF 4FDUSBOTMPDBTF XBT QSPWJEFE CZ UIF crystal structure of the SecA-SecYEG complex of Thermotoga maritima<>5IFTUSVDUVSF JOEJDBUFTUIBU4FD"FOUFSTUIF4FD:&(DIBOOFMCZNFBOTPGUIFTPDBMMFEUXPIFMJYmOHFS )' EPNBJOXIJDIDPOUBDUTUIFDZUPQMBTNJDMPPQPG4FD:*UIBTCFFOTVHHFTUFEUIBU UIF)'EPNBJOPG4FD"QMBZTBNBKPSSPMFJOQSPUFJOUSBOTMPDBUJPO5IF)'EPNBJOPG SecA may act as an arm that pushes the protein through the channel by means of a power. 95. 5.
(5) Chapter 5. strokeNFDIBOJTN< > BOEDSPTTMJOLJOHEBUBTVHHFTUTUIBUUIF)'JOUFSBDUTXJUIUIF USBOTMPDBUJOHQSFQSPUFJO"OBMUFSOBUJWFIZQPUIFTJTJTUIBUNPWFNFOUPGUIF)'USJHHFST channel opening and allows preprotein translocation by a Brownian ratchet mechanism < >*NQPSUBOUMZ TPGBSBOBDUVBMNPWFNFOUPGUIF)'IBTOPUCFFOEFNPOTUSBUFE In chapter 2 XFFYBNJOFEUIFJNQPSUBODFPGUIF)'4FD"JOQSPUFJOUSBOTMPDBUJPO CZFYUFOEJOHBOETIPSUFOJOHUIFMFOHUIPGUIF)'GPSVQUPBCPVUUXPIFMJDBMUVSOT )FSFJO CPUI IFMJDFT XIFSF FYUFOEFE BOE TIPSUFOFE UP MFBWF UIF UJQ PG UIF )' VOBMUFSFE5IFGVODUJPOBMJUZPGBTFUPG)'NVUBOUTPG4FD"XBTUFTUFEin vivo by a HSPXUI DPNQMFNFOUBUJPO BTTBZ PG B 4FD" UFNQFSBUVSF TFOTJUJWF TUSBJO 5IF EBUB TIPXFEDPNQMFUFGVODUJPOBMJUZPGUIF4FD"WBSJBOUXJUIBTZNNFUSJDBMMZTIPSUFE)' CVU UIF FYUFOEFE )' NVUBOUT XFSF VOBCMF UP DPNQMFNFOU UIF HSPXUI EFGFDU 5IF 4FD"NVUBOUTXJUIBOFYUFOEFE 4FD"J BOETIPSUFOFE 4FD"å )'XFSFGVSUIFS BOBMZTFEJOCJPDIFNJDBMBTTBZT*OMJOFXJUIUIFin vivo complementation assay result, 4FD"JXBTGPVOEUPCFJOBDUJWFXIJMF4FD"åTIPXFEBOFMFWBUFEUSBOTMPDBUJPO"51BTF BDUJWJUZCVUBSFEVDFEUSBOTMPDBUJPOFGmDJFODZBTDPNQBSFEUPXJMEUZQF4FD"6TJOH .JDSPTDBMF5IFSNPQIPSFTJT .45 XFPCTFSWFEUIBU4FD"åCJOETUP4FD:&(JOIJHI BGmOJUZ XIJMF 4FD"J BQQFBST NJTGPMEFE 5IFTF EBUB EFNPOTUSBUF UIBU UIF MFOHUI PG )'EPNBJOPG4FD"JTJNQPSUBOUGPSGVODUJPOJOHBT4FD"åSFRVJSFENPSFFOFSHZGPS USBOTMPDBUJPOQSPDFTT"QPTTJCMFFYQMBOBUJPOJTUIBU BTTVNJOHUIF)'BDUTBTBMFWFS UIFUSBOTMPDBUJPOTUFQTJ[FTHFUTTNBMMFSSFRVJSJOHNPSF"51UPUSBOTMPDBUFUIFQSPUFJO "MUFSOBUJWFMZ POFNBZBSHVFUIBU4FD"åJTMFTTFGmDJFOUJOPQFOJOHUIFUSBOTMPDBUJPO DIBOOFM UIBUNBZMFBEUPBOFOIBODFEFOFSHFUJDEFNBOEGPSUSBOTMPDBUJPO5IFSFGPSF UIFEBUBEPOPUEJGGFSFOUJBUFCFUXFFOUIFQPXFSTUSPLFPSNPMFDVMBSSBUDIFUNFDIBOJTN CVUFNQIBTJ[FUIFJNQPSUBODFPGUIF)'BTBGVODUJPOBMEPNBJOPG4FD" 4FWFSBMTUVEJFTIBWFCFFOEFNPOTUSBUFEUIBUUIF4FD:&(QPSFJTIJHIMZnFYJCMFBOE SFMBUJWFMZ OPOEJTDSJNJOBUJWF GPS QPMZQFQUJEF DPNQPTJUJPO 5IF QPSF FWFO DPOEVDUT UIF USBOTMPDBUJPO PG QSFQSPUFJOT DSPTTMJOLFE XJUI B OPOQFQUJEF DPOTUJUVFOUT < > Additionally, the SecYEG pore supports the translocation of proOmpA derivatives that BSFMBCFMFEXJUIUXPCVMLZnVPSPQIPSFBUUBDIFEBUUIF$UFSNJOVT<>*Ochapter 3, we further examined the permissiveness of the Sec translocase for the translocation PGDIFNJDBMMZNPEJmFEEFSJWBUJWFTPGQSP0NQ"XJUIBGPDVTPOUIFTJUFPGDIFNJDBM NPEJmDBUJPO'MVPSFTDFJONBMFJNJEF 'NBM BOEUIFQPMZNFSJDIZESPQIJMJDPMJHPFUIZMFOF glycol with two different contour length chains (malPEG1000 and malPEG3000) were JOUSPEVDFEBUDZTUFJOFQPTJUJPOTJOUIF/UFSNJOVTPGUIFNBUVSFSFHJPOPGQSP0NQ" 8IFSFBTMBCFMJOHBUUIF$UFSNJOVTIBTMJUUMFJNQBDUPOUIFUSBOTMPDBUJPOPGUIFQSP0NQ" EFSJWBUJWFT BUUBDINFOU PG nVPSPQIPSFT XJUIJO UIF mSTU BNJOP BDJET SFTJEVFT PG. 96.
(6) Summary. UIFNBUVSFSFHJPOPGQSP0NQ"ESBNBUJDBMMZSFEVDFUIFUSBOTMPDBUJPOFGmDJFODZ5IJT translocation defect was restored by the PrlA4 mutant of SecY which is believed to be FRVJQQFEXJUIBNPSFSFMBYFEQPSF<>-JLFXJTF QSP0NQ"EFSJWBUJWFTDPOKVHBUFEUP the polyethyleneglycol polymers normally translocated when the molecule was attached UP$UFSNJOBMSFTJEVFT)PXFWFS UIFTFTUSVDUVSFTIJOEFSFEUSBOTMPDBUJPOXIFOQSFTFOU in the extreme N-terminus of the mature region of proOmpA, a process that could not be SFTUPSFECZUIF1SM"NVUBOU1SFTVNBCMZ UIFTFCVMLZNPMFDVMFTJOIJCJUUSBOTMPDBUJPOCZ TUFSJDBMIJOEFSBODFQFSUVSCJOHUIFUSBOTMPDBUJPOJOJUJBUJPOQSPDFTT< >0WFSBMM UIFTF mOEJOHTEFNPOTUSBUFUIBU4FDUSBOTMPDBTFJTQFSNJTTJWFUPQSPUFJONPEJmDBUJPOPODFUIF USBOTMPDBUJPOIBTCFFOTVDDFTTGVMMZJOJUJBUFE)PXFWFS UIFEBUBBMTPTIPXTBSFNBSLBCMF MBDLPGTQFDJmDJUZPGUIF4FDUSBOTMPDBTFXIFSFCZCVMLZOPOQFQUJEFDPOTJTUFOUTDP USBOTMPDBUFXJUIQSP0NQ"CZBQJHHZCBDLNFDIBOJTN In chapter 4 UIFEZOBNJDTPGUIFJOUFSBDUJPOCFUXFFOUIF)'PG4FD"BOEUIF4FD:&( QPSFXBTJOWFTUJHBUFE*OQBSUJDVMBS UIJTDIBQUFSBEESFTTFTUIFRVFTUJPOBTUPXIFUIFSUIF )'PG4FD"NPWFTEVSJOHQSPUFJOUSBOTMPDBUJPO"TFUPGTJOHMFDZTUFJOF4FD"NVUBOUT XBTDSFBUFEUIBUXFSFMBCFMFEXJUIBEPOPSnVPSPQIPSF"MTP TJOHMFDZTUFJOFTNVUBUJPOT were introduced into the cysteine-less SecY, on cis water interface of TM2b, TM3 and 5.BOEUIFTFXFSFMBCFMFEXJUIBOBDDFQUPSnVPSPQIPSFUPDSFBUFTVJUBCMF'3&5QBJST XIFO4FD"BOE4FD:&(BSFCSPVHIUUPHFUIFS 5IF)'JTBOBDUJWFSFHJPOPGUIF4FD"QSPUFJO BOEUIVTJUTNVUBHFOFTJTBOEnVPSFTDFOU MBCFMJOHNBZQPUFOUJBMMZJOUFSGFSFXJUIBDUJWJUZ5IFSFGPSF in vitro translocation assays XFSFQFSGPSNFE TIPXJOHUIBUUIF4FD")'NVUBOUT,$BOE2$BSFTUJMMBDUJWF BGUFSQVSJmDBUJPOBOEMBCFMJOHXJUI*"/#%)PXFWFS TFWFSBMPUIFS4FD"NVUBOUTXFSF severely compromised in their activity whereas labeling with Cy3 also inactivated the 4FD")'NVUBOUT5IFDZTUFJOFNVUBUJPOTJO4FD:EJEOPUJOUFSGFSFXJUIBDUJWJUZBOE BMM5FYBT3FEMBCFMFE4FD:DZTUFJOFNVUBOUTXFSFBDUJWFJOUSBOTMPDBUJPO5IJTBOBMZTJT re-enforces the need to analyze the activity of the mutated proteins after labeling as to FYDMVEFUIPTFDPNCJOBUJPOTUIBUBSFTFWFSFMZJNQBJSFEJOBDUJWJUZ#BTFEPOUIFTFSFTVMUT '3&5FYQFSJNFOUTXFSFDPOEVDUFECZDPNCJOJOH*"/#%MBCFMFE4FD"QSPUFJOTXJUI JOOFSNFNCSBOFWFTJDMFTDPOUBJOJOHPWFSFYQSFTTFEMFWFMTPGUIF5FYBT3FEMBCFMFE4FD: '3&5TJHOBMXFSFPCTFSWFEXIFOEPOPSnVPSPQIPSFDPOKVHBUFE4FD"XBTBEEFEUPUIF BDDFQUPSDPOKVHBUFE4FD:&(DPOTJTUFOUXJUIBCJOEJOHFWFOU4JODFUIF'3&5TJHOBM XBTRVFODIFECZUIFBEEJUJPOPGVOMBCFMFE4FD" XFJOUFSQSFUFEUIBUUIF'3&5TJHOBMJT TQFDJmDGPSUIF4FD"4FD:&(CPVOETUBUF/PBMUFSFE'3&5TJHOBMXBTPCTFSWFEXIFO OVDMFPUJEFTXFSFBEEFEXJUIBOEXJUIPVUQSFQSPUFJOTVCTUSBUF5IJTmOEJOHTVHHFTUTOP GVSUIFSNBKPSDPOGPSNBUJPOBMDIBOHFTJOUIF)'SFHJPOPODF4FD"JTCPVOEUP4FD:&(. 97. 5.
(7) Chapter 5. 5IVT UIF'3&5BTTBZ IPXFWFS EPFTOPUTVQQPSUUIFOPUJPOUIBUUIF)'GVODUJPOTBT BO"51JOEVDFEMFWFSUIBUXPVMEESJWFUSBOTMPDBUJPOPSDIBOOFMPQFOJOH0OUIFPUIFS IBOE UIF)'NBZIBWFNPWFEPSDIBOHFEJUTDPOGPSNBUJPOEVSJOHCJOEJOHPG4FD"UP 4FD:&( CVUDBOOPUCFEFUFDUFEXJUIUIF'3&5BTTBZ Concluding remarks and future perspective SecA is a molecular motor that drives protein translocation by a conversion of chemical energy into a directional movement of the preprotein into the translocation channel <>)FSFXFGPVOEUIBU4FD:&(IBTBSFNBSLBCMFTVCTUSBUFQSPNJTDVJUZTJODFJUDBO USBOTMPDBUF MPOH TUSFUDIFT PG IZESPQIJMJD QPMZNFST 5IFTF CVMLZ HSPVQT IPXFWFS USBOTMPDBUF CZ NFBOT PG B QJHHZ CBDL NFDIBOJTN XJUI UIF QSFQSPUFJO $IFNJDBM NPEJm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binding may induce channel opening or widening of the channel which is not detected CZUIF'3&5BTTBZUIBUJOEJDBUFTOPDIBOHFJOEJTUBODFCFUXFFOUIF4FD")'BOE 4FD:'VSUIFSBOBMZTJTPGUIFNPWFNFOUTPG)'PG4FD"XJMMCFFTTFOUJBMEVFUPPVS VOEFSTUBOEJOHPGUIFNFDIBOJTNPGQSPUFJOUSBOTMPDBUJPO1PTTJCMZ '3&5BTTBZXJUI BDDFQUPSnVPSPQIPSFTBUPUIFSSFHJPOTPG4FD:NBZVONBTLBNPSFEZOBNJDCFIBWJPS UIBUNBZJODMVEFTJOHMFNPMFDVMFEFUFDUJPOPGUIF'3&5UPBDDPVOUGPSIFUFSPHFOFJUZPG MBCFMJOHBOEGVODUJPOBMJUZJOBCVMLQSPUFJOTBNQMF. 98.
(8) Summary. REFERENCES 7FFOFOEBBM",+ WBOEFS%PFT$%SJFTTFO"+. 5IFQSPUFJODPOEVDUJOHDIBOOFM 4FD:&(Biochim. Biophys. Acta - Mol. Cell Res. 1694 o %SJFTTFO"+./PVXFO/ 1SPUFJO5SBOTMPDBUJPO"DSPTTUIF#BDUFSJBM$ZUPQMBTNJD .FNCSBOFAnnu. Rev. Biochem. 77 o #FSH#WBOEFO $MFNPOT8. $PMMJOTPO* .PEJT: )BSUNBOO& )BSSJTPO4$3BQPQPSU5" 9SBZTUSVDUVSFPGBQSPUFJODPOEVDUJOHDIBOOFMNature 427 o ,VTUFST*%SJFTTFO"+. 4FD" BSFNBSLBCMFOBOPNBDIJOFCell. Mol. Life Sci. 68, o 7BO%FS8PML+18 %F8JU+(%SJFTTFO" 5IFDBUBMZUJDDZDMFPGUIFEscherichia coli 4FD""51BTFDPNQSJTFTUXPEJTUJODUQSFQSPUFJOUSBOTMPDBUJPOFWFOUTEMBO J. 16 o 4DIJFCFM& %SJFTTFO"+. )BSUM'68JDLOFS8 ϯ)
(9) BOE"51GVODUJPOBUEJGGFSFOU TUFQTPGUIFDBUBMZUJDDZDMFPGQSFQSPUFJOUSBOTMPDBTFCell 64 o 5TVLB[BLJ5/VSFLJ0 5IFNFDIBOJTNPGQSPUFJOFYQPSUFOIBODFNFOUCZUIF4FD%' NFNCSBOFDPNQPOFOUBiophysics (Oxf). 7 o #FDIUMVGU1 /PVXFO/ 5BOT4+%SJFTTFO"+. 4FD#BDIBQFSPOFEFEJDBUFEUP QSPUFJOUSBOTMPDBUJPOMol. Biosyst. 6 o /JTIJZBNB,*5PLVEB) 1SFQBSBUJPOPGBIJHIMZUSBOTMPDBUJPODPNQFUFOUQSP0NQ" 4FD#DPNQMFYProtein Sci. 19 o ,PDI)( )FOHFMBHF5 /FVNBOO)BFGFMJO$ .BD'BSMBOF+ )PGGTDIVMUF), 4DIJN[,- .FDIMFS#.àMMFS. In vitroTUVEJFTXJUIQVSJmFEDPNQPOFOUTSFWFBMTJHOBMSFDPHOJUJPO QBSUJDMF 431 BOE4FD"4FD#BTDPOTUJUVFOUTPGUXPJOEFQFOEFOUQSPUFJOUBSHFUJOHQBUIXBZT PG&TDIFSJDIJBDPMJMol. Biol. Cell 10 o )FOOPO48 4PNBO3 ;IV-%BMCFZ3& :JE$"MC0YBGBNJMZPGJOTFSUBTFTJ. Biol. Chem. 290 ;JNNFS+ /BN:3BQPQPSU5" 4USVDUVSFPGBDPNQMFYPGUIF"51BTF4FD"BOEUIF QSPUFJOUSBOTMPDBUJPODIBOOFMNature 455 o 6DIJEB, .PSJ).J[VTIJNB4 4UFQXJTFNPWFNFOUPGQSFQSPUFJOTJOUIFQSPDFTT of translocation across the cytoplasmic membrane of Escherichia coliJ. Biol. Chem. 270, o 4JNPO4. 1FTLJO$40TUFS(' 8IBUESJWFTUIFUSBOTMPDBUJPOPGQSPUFJOT Proc. Natl. Acad. Sci. U. S. A. 89 o "MMFO8+ $PSFZ3" 0BUMFZ1 4FTTJPOT3# 3BEGPSE4& 5VNB3$PMMJOTPO* 5XP way communication between SecY and SecA suggests a Brownian ratchet mechanism for QSPUFJOUSBOTMPDBUJPOElife 5 ,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. 99. 5.
(10) Chapter 5. ,FZ[FS+%F %PFT$7BO%FS%SJFTTFO"+. ,JOFUJD"OBMZTJTPGUIF5SBOTMPDBUJPOPG Fluorescent Precursor Proteins into Escherichia coli.FNCSBOF7FTJDMFT 277 o EF,FZ[FS+ WBOEFS%PFT$ 4XBWJOH+%SJFTTFO"+. 5IF':NVUBUJPOPG1SM" UFNQFSTUIFTJHOBMTFRVFODFTVQQSFTTPSQIFOPUZQFCZSFEVDJOHUIF4FD"CJOEJOHBGmOJUZ FEBS Lett. 510 o 5TJSJHPUBLJ " $IBU[J ,& ,PVLBLJ . %F (FZUFS + 1PSUBMJPV "( 0SGBOPVEBLJ ( 4BSEJT .' 5SFMMF .# +SHFOTFO 5+% ,BSBNBOPV 4 &DPOPNPV " -POH-JWFE 'PMEJOH *OUFSNFEJBUFT1SFEPNJOBUFUIF5BSHFUJOH$PNQFUFOU4FDSFUPNFStructure 26 oF 5PNLJFXJD[% /PVXFO/%SJFTTFO"+. 1VTIJOH QVMMJOHBOEUSBQQJOHo.PEFTPG NPUPSQSPUFJOTVQQPSUFEQSPUFJOUSBOTMPDBUJPOFEBS Lett. 581 o. 100.
(11) Summary. SAMENVATTING $FMMVMBJSFQSPDFTTFOCFUSFGGFOOJFUBMMFFOEFBDUJWJUFJUWBOFJXJUUFO NBBSPPLPOUFMCBSF JOUFSBDUJFT UVTTFO FJXJUUFO *O CBDUFSJÑO XPSEFO EF FJXJUUFO HFTZOUIFUJTFFSE EPPS SJCPTPNFOEJFHFMFHFO[JKOJOIFUDZUPTPM&DIUFS FFOTVCTUBOUJFFMHFEFFMUFWBOEF FJXJUUFOIFFGUFFOGVODUJFCVJUFOIFUDZUPTPM#JKWPPSCFFMEJOEscherichia coli wordt een deel van de cellulaire eiwitten over het cytoplasmatisch membraan getransloceerd of in EJUNFNCSBBOHFÕOUFHSFFSE)FUTZTUFFNEBUWFSBOUXPPSEFMJKLJTWPPSEJUQSPDFTJTEF 4FDSPVUF< >%F[FSPVUFCFTUBBUVJUIFU4FDUSBOTMPDBTF CFTUBBOEFVJUFFO4FD:&( IFUFSPUSJNFFSNFNCSBBOFJXJUDPNQMFY EBUFFOFJXJUHFMFJEFOELBOBBMJOIFUNFNCSBBO WPSNU<> IFUNPUPSFJXJU4FD"EBUNJEEFMT"51IZESPMZTFEFUSBOTMPDBUJFBBOESJKGU<o> FOIFUCJKLPNFOEFIFUFSPEJNFFSNFNCSBBOFJXJUDPNQMFY4FD%'ZBK$EBUUSBOTMPDBUJF LPQQFMUBBOEFQSPUPOESJKWFOEFLSBDIUPGIFUNFNCSBBO<> 7PPSIFUMPLBMJTFSFOWBOFJXJUUFOOBBSIFU4FDUSBOTMPDBTF CFTUBBOFSUXFFWFSTDIJMMFOEF XFHFO &JXJUUFO EJF NPFUFO XPSEFO VJUHFTDIFJEFO XPSEFO HFTZOUIFUJTFFSE NFU een signaal-sequentie op de N-terminus dat ze in een ongevouwen staat naar het SecYEG-gebonden SecA aan de membraan stuurt, dan wel met of zonder hulp van de NPMFDVMBJSFDIBQFSPOOF4FD#< >%JUNFDIBOJTNFXPSEUPPLXFMEFQPTUUSBOTMBUJPOFMF SPVUF HFOPFNE BBOHF[JFO EF USBOTMPDBUJF WBO EF[F FJXJUUFO OJFU HFLPQQFME JT BBO QPMZQFQUJEFLFUFOWFSMFOHJOH PQ IFU SJCPTPPN $ZUPQMBTNBUJTDIF NFNCSBBOFJXJUUFO XPSEFO IFSLFOE BBO IVO FFSTUF IZESPGPCJTDIF USBOTNFNCSBBO TFHNFOU UJKEFOT de translatie aan het ribosoom en worden vervolgens gebonden door het signaalIFSLFOOJOHTEFFMUKF TJHOBMSFDPHOJUJPOQBSUJDMF 431 %JUESJFWPVEJHDPNQMFY HFWPSNE EPPS IFU SJCPTPPN 431 FO IFU EFFMT HFTZOUIFUJTFFSEF NFNCSBBOFJXJU OBTDFOU membrane protein), wordt vervolgens co-translationeel naar het membraan gebracht FOCJOEUEBBSEF431SFDFQUPS'UT:<>7FSWPMHFOT[PSHUEFCJOEJOHFOIZESPMZTF WBO (51 EPPS IFU IFUFSPEJNFFS 431'UT: DPNQMFY WPPS IFU WFSQMBBUTFO WBO IFU deels gesynthetiseerde membraaneiwit naar het SecYEG complex, dat het nieuwe HFTZOUIFUJTFFSEFNFNCSBBOFJXJUWFSWPMHFOTJOUFHSFFSUJOIFUNFNCSBBN%JUQSPDFTJT HFLPQQFMEBBOQPMZQFQUJEFLFUFOWFSMFOHJOHBBOIFUSJCPTPPN:JE$LBO4FD:&(IFMQFO CJKIFUJOTFSUFSFOWBONFNCSBBOFJXJUUFO<>#FJEFSPVUFT[JKOHFEVSFOEFEFMBBUTUF decennia uitgebreid bestudeerd met behulp van genetische, biochemische en structurele NFUIPEFO0OTIVJEJHCFHSJQWBOEFGVODUJPOFMFFOTUSVDUVSFMFFJHFOTDIBQQFOWBO 4FD:&(FO4FD"[JKOTBNFOHFWBUJOhoofdstuk 1WBOEJUQSPFGTDISJGU &FOCFMBOHSJKLFWPPSVJUHBOHJOIFUCFHSJKQFOWBOIFU4FDUSBOTMPDBTF XBTNPHFMJKL NFUEFLSJTUBMTUSVDUVVSWBOIFU4FD"4FD:&(DPNQMFYVJUThermotoga maritima <>. 101. 5.
(12) Chapter 5. %FTUSVDUVVSHFFGUBBOEBU4FD"IFU4FD:&(LBOBBMCJOOFOLPNUEPPSNJEEFMWBOFFO [PHFOBBNEFUXFFIFMJYWJOHFS )' EJFJODPOUBDUTUBBUNFUEFFDZUPQMBTNBUJTDIF MVTWBO4FD:&SXBTHFTVHHFSFFSEEBUIFU)'EPNFJOFFOCFMBOHSJKLFSPMTQFFMUJO FJXJUUSBOTMPDBUJF)FU)'EPNFJOWBO4FD"LBO[JDINPHFMJKLBMTFFOBSNHFESBHFO EBUIFUFJXJUEPPSIFULBOBBMLBOESVLLFO EPPSNJEEFMWBOFFOLSBDIUTMBHNFDIBOJTNF < >7FSEFSTVHHFSFSFODIFNJTDIFDSPTTMJOLJOHFYQFSJNFOUFOEBUEF)'JOUFSBDUFFSU NFUIFUUSBOTMPDFSFOEFFJXJU&FOBMUFSOBUJFWFIZQPUIFTFJTEBUEFCFXFHJOHWBOEF )' FFO PQFOJOH JO IFU LBOBBM WFSPPS[BBLU XBBSEPPS IFU FJXJU TVCTUSBBU EPPS IFU LBOBBM USBOTMPDFFSU NJEEFMT FFO NPMFDVMBJS SBUFM #SPXOJBO SBUDIFU NFDIBOJTNF < >#FMBOHSJKLPNUFWFSNFMEFOJTEBUXFSLFMJKLFCFXFHJOHWBOEF)'OPHOPPJUJT HFEFNPOTUSFFSE In hoofdstuk 2JTIFUCFMBOHWBOEF)'4FD"JOFJXJUUSBOTMPDBUJFPOEFS[PDIUEPPSEF[F UFWFSMFOHFOFOWFSLPSUFONFUNBYJNBBMPOHFWFFSUXFFIFMJYPNXFOUFMJOHFO)JFSJO XBSFOCFJEFIFMJDFTWFSMFOHEPGWFSLPSU [PEBUIFUVJUFJOEFWBOEF)'POBBOHFQBTU CMFFG%FGVODUJPOBMJUFJUWBOFFOSFFLTWBO)'NVUBOUFOWBO4FD"JTHFUFTUJOWJWP door middel van een groei-complementatie analyse van een SecA temperatuurgevoelige TUBN%F4FD"WBSJBOUNFUFFOTZNNFUSJTDIWFSLPSUF)'CMFFLGVODUJPOFFM FDIUFSEF WFSMFOHEF)'NVUBOUFOXBSFOOJFUJOTUBBUPNIFUHSPFJEFGFDUUFDPNQMFNFOUFSFO %F4FD"NVUBOUFONFUFFOWFSMFOHEF 4FD"J FOWFSLPSUF 4FD"å )'[JKOWFSEFS HFBOBMZTFFSEJOCJPDIFNJTDIFFYQFSJNFOUFO*OPWFSFFOTUFNNJOHNFUEFJOWJWPHSPFJ DPNQMFNFOUBUJFBOBMZTF XFSE4FD"JJOBDUJFGCFWPOEFOUFSXJKM4FD"åFFOWFSIPPHEF USBOTMPDBUJF "51BTFBDUJWJUFJU NBBS FFO WFSMBBHEF USBOTMPDBUJFFGmDJÑOUJF WFSHFMFLFO NFU IFU PPSTQSPOLFMJKLF 4FD" FJXJU .JEEFMT NJDSPTDIBBM UIFSNPGPSFTF Microscale Thermophoresis .45 OBNFOXFXBBSEBU4FD"å4FD:&(NFUFFOIPHFBGmOJUFJU CJOEUUFSXJKM4FD"JWFSLFFSEHFWPVXFOMJKLUUF[JKO%F[FHFHFWFOTUPOFOBBOEBUEF MFOHUFWBOIFU)'EPNFJOWBO4FD"CFMBOHSJKLJTWPPSIFUGVODUJPOFSFO BBOHF[JFO 4FD"åNFFSFOFSHJFWPPSUSBOTMPDBUJFOPEJHIFFGU&FONPHFMJKLFWFSLMBSJOHJTEBU BBOOFNFOEFEBU)'BMTFFOIFGCPPNGVODUJPOFFSU EFUSBOTMPDBUJFTUBQHSPPUUFLMFJOFS XPSEUXBBSEPPSNFFS"51OPEJHJTPNIFUFJXJUUFUSBOTMPDFSFO&FOBMUFSOBUJFGJTEBU 4FD"åNJOEFSFGmDJÑOUJTJOIFUPQFOFOWBOIFUUSBOTMPDBUJFLBOBBM XBUFFOWFSIPPHE FOFSHFUJTDIWFSFJTUFWPPSUSBOTMPDBUJFWFSPPS[BBLU%FSIBMWFEJGGFSFOUJÑSFOEF[FHFHFWFOT OJFUUVTTFOIFULSBDIUTMBHFONPMFDVMBJSFSBUDIFUNPEFM NBBSCFOBESVLLFOIFUCFMBOH WBOEF)%BMTFFOGVODUJPOFFMEPNFJOWBO4FD" 7FSTDIJMMFOEFTUVEJFTIFCCFOBBOHFUPPOEEBUEF4FD:&(QPSJF[FFSnFYJCFMFOSFMBUJFG OJFU EJTDSJNJOFSFOE JT WPPS EF BNJOP[VVSTBNFOTUFMMJOH WBO IFU TVCTUSBBU %F QPSJF HFMFJEU [FMGT EF USBOTMPDBUJF WBO FJXJUUFO WFSLOPPQU NFU OJFUQFQUJEF CFTUBOETEFMFO. 102.
(13) Summary. < >%F4FD:&(QPSJFPOEFSTUFVOUPPLEFUSBOTMPDBUJFWBOQSP0NQ"EFSJWBUFOEJF HFMBCFME[JKONFUUXFFPNWBOHSJKLFnVPSPGPSFOBBOEF$UFSNJOVT<>*Ohoofdstuk 3 hebben we de tolerantie van de Sec translocase voor de translocatie van chemisch HFNPEJmDFFSEFEFSJWBUFOWBOQSP0NQ"WFSEFSPOEFS[PDIU NFUFFOGPDVTPQEFQMBBUT WBODIFNJTDIFNPEJmDBUJF'MVPSFTDFÕOFNBMFÕNJEF 'NBM FOIFUQPMZNFSFIZESPmFMF oligo-ethyleen glycol met twee verschillende contour lengtes (malPEG1000 and malPEG3000) [JKOHFÕOUSPEVDFFSEPQEFDZTUFÕOFQPTJUJFTJOEF/UFSNJOVTWBOEFHFNBUVSFFSEFSFHJP WBOQSP0NQ")FUJOCSFOHFOWBOnVPSPGPSFOPQEFFFSTUFBNJOP[VVSSFTJEVFOWBO de gematureerde regio van proOmpA resulteerde in een dramatische verlaging van EFUSBOTMPDBUJFFGmDJÑOUJF*OEJFOEFDIFNJTDIFNPEJmDBUJFTJOEF$UFSNJOVTXFSEFO BBOHFCSBDIU CMFFLEJUXFJOJHJOWMPFEPQEFUSBOTMPDBUJFWBOQSP0NQ"EFSJWBUFOUF IFCCFO)FUFFSEFSHFOPFNEFUSBOTMPDBUJFEFGFDULPOXPSEFOIFSTUFMEEPPSEF1MS" NVUBOU WBO 4FD: UF HFCSVJLFO EJF WFSPOEFSTUFME XPSEU FFO NFFS POUTQBOOFO QPSJF UF CF[JUUFO <> 0PL JO IFU HFWBM WBO EF QSP0NQ" EFSJWBUFO HFDPOKVHFFSE BBO EF QPMZFUIZMFFOHMZDPM QPMZNFSFO CMFFL USBOTMPDBUJF OPSNBBM XBOOFFS EF[F HFLPQQFME XBSFOBBOEF$UFSNJOBMFSFTJEVFO&DIUFS EF[FTUSVDUVSFOCMPLLFFSEFOUSBOTMPDBUJF wanneer aanwezig in de extreme N-terminus van de gematureerde regio van proOmpA, FFOQSPDFTEBUOJFUIFSTUFMELPOXPSEFOEPPSEF1SM"NVUBOU8BBSTDIJKOMJKLSFNNFO EF[FPNWBOHSJKLFNPMFDVMFOUSBOTMPDBUJFEPPSNJEEFMWBOTUFSJTDIFIJOEFSJOHEBUIFU QSPDFTWBOUSBOTMPDBUJFJOJUJBUJFWFSTUPPSU XBBSCJKFFOIFMJTDIFIBBSTQFMEHFWPSNENPFU worden tussen de signaal peptide en het N-terminal mature domein van het translocatie FJXJU< >%F[FXBBSOFNJOHFOUPOFOBBOEBUIFU4FDUSBOTMPDBTF[POEFSQSPCMFNFO BMMFSMFJDIFNJTDIFNPEJmDBUJFTWBOIFUFJXJUTVCTUSBBUUPFTUBBU NJUTUSBOTMPDBUJFTVDDFTWPM HFÕOJUJFFSE LBO XPSEFO 8BBSTDIJKOMJKL XPSEFO EF OJFUQFQUJEF CFTUBOETEFMFO NFU QSP0NQ"HFUSBOTMPDFFSEEPPSNJEEFMWBOFFOQJHHZCBDLNFDIBOJTNF In hoofdstuk 4[JKOEFEZOBNJFLFOWBOEFJOUFSBDUJFTUVTTFOWB)'WBO4FD"FOEF 4FD:&(QPSJFPOEFS[PDIU*OIFUCJK[POEFSSJDIUEJUIPPGETUVL[JDIPQEFWSBBHPGEF )'WBO4FD"CFXFFHUUJKEFOTFJXJUUSBOTMPDBUJF)JFSUPFJTFFOSFFLTWBOFOLFMWPVEJHF DZTUFÕOFNVUBOUFOWBOIFUDZTUFÕOFWSJKF4FD"HFNBBLU EJFWFSWPMHFOTHFMBCFME[JKO NFUFFOEPOPSnVPSPGPPS$ZTUFÕOFT[JKOPPLHFÕOUSPEVDFFSEPQIFUHSFOTWMBLWBOXBUFS NFNCSBBOJO5.C 5.FO5.WBO4FD:%F[FXFSEFOHFMBCFMENFUFFOnVPSPGPPS BDDFQUBOUPN[PHFOBBNEF'3&5QBSFOUFWPSNFOXBOOFFS4FD"FO4FD:&(UF[BNFO LPNFO%F)'JTFFOBDUJFWFSFHJPWBOIFU4FD"FJXJU XBBSEPPSEFNVUBHFOFTFFO nVPSFTDFOUFMBCFMJOHNPHFMJKLJOWMPFEIFFGUPQEFBDUJWJUFJU%BBSPNXFSEFOJOWJUSP USBOTMPDBUJF BOBMZTFT HFEBBO EJF MJFUFO [JFO EBU EF 4FD" )' NVUBOUFO ,$ FO 2$OPHTUFFETBDUJFG[JKOOB[VJWFSJOHFOMBCFMJOHNFU*"/#%%BBSFOUFHFOXFSEFO WFSTDIJMMFOEFBOEFSF4FD"TUFSLHFIJOEFSEJOIVOBDUJWJUFJUFOPPLMBCFMJOHNFU$Z. 103. 5.
(14) Chapter 5. IBEFFOJOBDUJWFSFOEFXFSLJOH%FDZTUFÕOFNVUBOUFOJO4FD:IBEEFOHFFOTUPSFOEF XFSLJOHNFUCFUSFLLJOHUPUEFBDUJWJUFJU FWFOBMTEF5FYBT3FEHFMBCFMEF4FD:DZTUFÕOF NVUBOUFOEJFBMMFOBDUJFGXBSFOJOUSBOTMPDBUJF%FBOBMZTFCFOBESVLUEFOPPE[BBLPN de activiteit van de gemuteerde eiwitten na labeling te meten om de combinaties die EFBDUJWJUFJUTUFSLWFSTUPSFOVJUUFTMVJUFO(FCBTFFSEPQEF[FSFTVMUBUFOXFSEFO'3&5 experimenten uitgevoerd, door het combineren van SecA gelabelde IANBD met binnenNFNCSBBOCMBBTKFT NFUEBBSJOUPUPWFSFYQSFTTJFHFCSBDIUF5FYBT3FEHFMBCFMEF4FD: '3&5TJHOBMFOXFSEFOXBBSHFOPNFOXBOOFFSEFEPOPSnVPSPGPPSHFDPOKVHFFSEF4FD" XFSEUPFHFWPFHEBBOEFBDDFQUPSHFDPOKVHFFSEF4FD:&( JOPWFSFFOTUFNNJOHNFUIFU GFJUEBUEF[FFJXJUUFOFFODPNQMFYWPSNFO%BBSIFU'3&5TJHOBBMHFEPPGEXFSEEPPS EFUPFWPFHJOHWBOPOHFMBCFMEF4FD" JTEJUTQFDJmFLWPPSEF4FD"4FD:&(HFCPOEFO TUBBU&SXFSEFOHFFOWFSBOEFSJOHFOJOEF'3&5TJHOBMFONFFSHFEFUFDUFFSEXBOOFFS OVDMFPUJEFOXFSEFOUPFHFWPFHE [PXFMJOBBOFOBGXF[JHIFJEWBOFFOTVCTUSBBUFJXJU Deze waarneming suggereert dat er geen verdere conformationele veranderingen in EF)'SFHJP[JKOOBEBU4FD"HFCPOEFOJTBBO4FD:&(%BBSPNPOEFSTUFVOUEF'3&5 BOBMZTFOJFUIFUNPEFMXBBSCJKEF)'GVODUJPOFFSUBMTFFO"51HFÕOEVDFFSEFIFGCPPN EBUEFUSBOTMPDBUJFBBOESJKGU"BOEFBOEFSFLBOULBOEF)'NPHFMJKLXFMBM[JKOCFXPHFO of veranderd op het moment dat de binding tussen SecA aan SecYEG plaatsvindt, maar EJUPOEFSTDIFJELPOOJFUXPSEFOHFNBBLUPQCBTJTWBOEF'3&5BOBMZTF Slotopmerkingen en toekomstperspectief SecA is een moleculair motoreiwit dat het proces van eiwit-translocatie over de DZUPQMBTNBUJTDIFNFNCSBBOBBOESJKGUEPPSNJEEFMWBOFFODPOWFSTJFWBODIFNJTDIF FOFSHJFJOFFOEJSFDUJPOFMFCFXFHJOHWBOIFUTVCTUSBBUFJXJUEPPSIFUUSBOTMPDBUJFLBOBBM <> 4FD:&( CMJKLU PQNFSLFMJKL XFJOJH POEFSTDIFJE UF NBLFO UVTTFO TVCTUSBUFO BBOHF[JFO IFU PPL OJFUFJXJU TFHNFOUFO LBO USBOTMPDFSFO [PBMT MBOH VJUHFTUSFLUF IZESPmFMFQPMZNFSFO&DIUFS EF[FPNWBOHSJKLFOJFUFJXJUHSPFQFOXPSEFOHFUSBOTMPDFFSU EPPSNJEEFMWBOFFOQJHHZCBDLNFDIBOJTNFTBNFONFUIFUTVCTUSBBUFJXJU$IFNJTDIF NPEJmDBUJFWBOEF/UFSNJOVTHFNBUVSFFSEFSFHJPWBOIFUTVCTUSBBUFJXJUJOUFSGFSFFSUTUFSL NFUEFUSBOTMPDBUJF XBUTVHHFSFFSUEBUJOEF[FSFHJPFFOTUSJLUFSFDPOGPSNBUJPOFMFFJT CFTUBBUEJFXBBSTDIJKOMJKLEFJOJUJÑMFWPSNJOHWBOEFIFMJTDIFIBBSTQFMEUVTTFOEFTJHOBBM TFRVFOUJF FO EF /UFSNJOVT HFNBUVSFFSEF SFHJP CFUSFGU %F DIFNJTDIF NPEJmDBUJF WPPSLPNU USBOTMPDBUJF XBBSTDIJKOMJKL EPPS TUFSJTDIF IJOEFSJOH XBBSEPPS EF IFMJTDIF IBBSTQFMEPGOJFUNFFSQBTUJOIFUUSBOTMPDBUJFLBOBBM PGOJFUHFWPSNELBOXPSEFO %F)'SFHJPWBO4FD"JOTFSFFSUJOEFDZUPQMBTNBUJTDIFPQFOJOHWBO4FD:XBBSCJK IFUUPQKFWBOEF)'EJDIUCJKIFUHFUSBOTMPDFFSEFTVCTUSBBUFJXJU[JU WMBLCJKEFJOHBOH WBOEF4FD:&(QPSJF<>&DIUFS IFUNFDIBOJTNFXBBSNFFEF)'CJKESBBHUBBO. 104.
(15) Summary. EF USBOTMPDBUJF JT OPH TUFFET POCFLFOE 7FSEFS JT IFU CFMBOHSJKL EBU FS HFFO HSPUF CFXFHJOHFOWBOEF)'UFOPQ[JDIUFWBOIFU4FD:LBOBBMXFSEFOHFEFUFDUFFSEOB CJOEJOHWBO"51BBO4FD" XBUWPPSFFOTUBQTHFXJK[FFJXJUUSBOTMPDBUJF[PSHU%JUTMVJU OJFUVJUEBU4FD"EFPQFOJOHPGIFUWFSXJKEFOWBOIFU4FD:LBOBBMJOEVDFFSU EBBSEJU BMQMBBUTLBOWJOEFOUJKEFOTEFCJOEJOHWBO4FD"BBO4FD:&(7FSEFSFBOBMZTFBBOEF CFXFHJOHWBO)'WBO4FD"JTFTTFOUJFFMWPPSPOTCFHSJQWBOIFUNFDIBOJTNFWBO FJXJUUSBOTMPDBUJF .PHFMJKL LBO FFO '3&5 BOBMZTF NFU EF BDDFQUFSFOEF nVPSGPPS PQ BOEFSFSFHJPTWBO4FD:FFONFFSEZOBNJTDIHFESBHJOLBBSUCSFOHFO%BBSCJKJTFOLFM NPMFDVVMEFUFDUJFWBOEF'3&5OPEJHPNSFLFOJOHUFIPVEFONFUEFIFUFSPHFOJUFJUWBO EFMBCFMJOHFOGVODUJPOBMJUFJUWBOEFFJXJUUFO. REFERENTIES 7FFOFOEBBM",+ WBOEFS%PFT$%SJFTTFO"+. 5IFQSPUFJODPOEVDUJOHDIBOOFM 4FD:&(Biochim. Biophys. Acta - Mol. Cell Res. 1694 o %SJFTTFO"+./PVXFO/ 1SPUFJO5SBOTMPDBUJPO"DSPTTUIF#BDUFSJBM$ZUPQMBTNJD .FNCSBOFAnnu. Rev. Biochem77 o #FSH#WBOEFO $MFNPOT8. $PMMJOTPO* .PEJT: )BSUNBOO& )BSSJTPO4$3BQPQPSU5" 9SBZTUSVDUVSFPGBQSPUFJODPOEVDUJOHDIBOOFM Nature 427 o ,VTUFST*%SJFTTFO"+. 4FD" BSFNBSLBCMFOBOPNBDIJOFCell. Mol. Life Sci. 68, o 7BO%FS8PML+18 %F8JU+(%SJFTTFO" 5IFDBUBMZUJDDZDMFPGUIFEscherichia coli 4FD""51BTFDPNQSJTFTUXPEJTUJODUQSFQSPUFJOUSBOTMPDBUJPOFWFOUT EMBO J. 16 o 4DIJFCFM& %SJFTTFO"+. )BSUM'68JDLOFS8 ϯ)
(16) BOE"51GVODUJPOBUEJGGFSFOU TUFQTPGUIFDBUBMZUJDDZDMFPGQSFQSPUFJOUSBOTMPDBTFCell 64 o 5TVLB[BLJ5/VSFLJ0 5IFNFDIBOJTNPGQSPUFJOFYQPSUFOIBODFNFOUCZUIF4FD%' NFNCSBOFDPNQPOFOUBiophysics (Oxf). 7 o #FDIUMVGU1 /PVXFO/ 5BOT4+%SJFTTFO"+. 4FD#BDIBQFSPOFEFEJDBUFEUP QSPUFJOUSBOTMPDBUJPOMol. Biosyst. 6 o /JTIJZBNB,*5PLVEB) 1SFQBSBUJPOPGBIJHIMZUSBOTMPDBUJPODPNQFUFOUQSP0NQ" 4FD#DPNQMFYProtein Sci. 19 o ,PDI)( )FOHFMBHF5 /FVNBOO)BFGFMJO$ .BD'BSMBOF+ )PGGTDIVMUF), 4DIJN[,- .FDIMFS#.àMMFS. In vitroTUVEJFTXJUIQVSJmFEDPNQPOFOUTSFWFBMTJHOBMSFDPHOJUJPO QBSUJDMF 431 BOE4FD"4FD#BTDPOTUJUVFOUTPGUXPJOEFQFOEFOUQSPUFJOUBSHFUJOHQBUIXBZT of Escherichia coli. Mol. Biol. Cell 10 o )FOOPO48 4PNBO3 ;IV-%BMCFZ3& :JE$"MC0YBGBNJMZPGJOTFSUBTFTJ. Biol. Chem. ;JNNFS+ /BN:3BQPQPSU5" 4USVDUVSFPGBDPNQMFYPGUIF"51BTF4FD"BOEUIF QSPUFJOUSBOTMPDBUJPODIBOOFMNature 455 o. 105. 5.
(17) Chapter 5. 6DIJEB, .PSJ).J[VTIJNB4 4UFQXJTFNPWFNFOUPGQSFQSPUFJOTJOUIFQSPDFTT of translocation across the cytoplasmic membrane of Escherichia coli. J. Biol. Chem. 270, o 4JNPO4. 1FTLJO$40TUFS(' 8IBUESJWFTUIFUSBOTMPDBUJPOPGQSPUFJOT Proc. Natl. Acad. Sci. U. S. A. 89 o "MMFO8+ $PSFZ3" 0BUMFZ1 4FTTJPOT3# 3BEGPSE4& 5VNB3$PMMJOTPO* 5XP way communication between SecY and SecA suggests a Brownian ratchet mechanism for QSPUFJOUSBOTMPDBUJPOElife ,BUP..J[VTIJNB4 5SBOTMPDBUJPOPGDPOKVHBUFEQSFTFDSFUPSZQSPUFJOTQPTTFTTJOH an internal non-peptide domain into everted membrane vesicles in Escherichia coli. J. 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$7BO%FS%SJFTTFO"+. ,JOFUJD"OBMZTJTPGUIF5SBOTMPDBUJPOPG Fluorescent Precursor Proteins into Escherichia coli.FNCSBOF7FTJDMFT 277 o EF,FZ[FS+ WBOEFS%PFT$ 4XBWJOH+%SJFTTFO"+. 5IF':NVUBUJPOPG1SM" UFNQFSTUIFTJHOBMTFRVFODFTVQQSFTTPSQIFOPUZQFCZSFEVDJOHUIF4FD"CJOEJOHBGmOJUZ FEBS Lett. 510 o 5TJSJHPUBLJ " $IBU[J ,& ,PVLBLJ . %F (FZUFS + 1PSUBMJPV "( 0SGBOPVEBLJ ( 4BSEJT .' 5SFMMF .# +SHFOTFO 5+% ,BSBNBOPV 4 &DPOPNPV " -POH-JWFE 'PMEJOH *OUFSNFEJBUFT1SFEPNJOBUFUIF5BSHFUJOH$PNQFUFOU4FDSFUPNF Structure 26 oF 5PNLJFXJD[% /PVXFO/%SJFTTFO"+. 1VTIJOH QVMMJOHBOEUSBQQJOHo.PEFTPG NPUPSQSPUFJOTVQQPSUFEQSPUFJOUSBOTMPDBUJPOFEBS Lett. 581 o. 106.
(18) Summary. RINGKASAN 1SPTFTQSPTFT EJ EBMBN TFM NFMJCBULBO BLUJWJUBTBLUJWJUBT QSPUFJO EBO KVHB JOUFSBLTJ QSPUFJO1BEBCBLUFSJ NJTBMOZBEscherichia coli, protein disintesis oleh ribosom yang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o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bab 1 LBNJNFSBOHLVN QFNBIBNBOUFSLJOJNFOHFOBJGVOHTJEBOLBSBLUFSJTUJLTUSVLUVSBM4FD:&(EBO4FD" 1FOFMJUJBOUFOUBOH4FDUSBOTMPLBTFCFSLFNCBOHQFTBUTFUFMBINVODVMOZBIBTJMQFOFOUVBO TUSVLUVSQSPUFJOLPNQMFLT4FD"4FD:&(Thermotoga maritima#FSEBTBSLBOTUSVLUVS LPNQMFLTQSPUFJO4FD"4FD:&( UFSMJIBUCBIXB4FD"NFOHJLBU4FD:&(NFMBMVJTFCVBI SFHJPOIFMJLTEVBKBSJ UXPIFMJYmOHFS )' )'4FD"CFSTJOHHVOHBOEFOHBOQVUBSBO MPPQTJUPQMBTNJLQSPUFJO4FD:TFIJOHHBEPNBJO)'4FD"EJVTVMLBONFNJMJLJQFSBO QFOUJOHEBMBNNFLBOJTNFUSBOTMPLBTJQSPUFJO5FSEBQBUEVBIJQPUFTJTQFSBO)'4FD" EBMBNNFLBOJTNFUSBOTMPLBTJQSPUFJO1FSUBNB EPNBJO)'4FD"CFSQFSBOTFPMBIPMBI. 107. 5.
(19) Chapter 5. TFQFSUJiMFOHBOwZBOHNFOEPSPOHQPMJQFQUJEBNBTVLLFEBMBNQPSJ4FD:&()BTJMVKJ CJPLJNJBNFOVOKVLLBOCBIXB)'CFSJOUFSBLTJEFOHBOQSFQSPUFJOUSBOTMPLBTJ,FEVB QFSHFSBLBO)'NFNJDVUFSCVLBOZBQPSJ4FD:&(EBOUSBOTMPLBTJQSFQSPUFJOUFSKBEJ EFOHBONFLBOJTNF#SPXOJBOSBUDIFU< >/BNVOTFKBVIJOJHFSBLBOBLUVBM)' CFMVNCFMVNEJLFUBIVJTFDBSBQFSTJT1BEBbab 2 LBNJNFNQFMBKBSJUFOUBOHQFOUJOHOZB EPNBJO)'4FD"EBMBNQSPTFTUSBOTMPLBTJQSPUFJOEFOHBODBSBSFLBZBTBHFOFUJLZBJUV NFNBOKBOHLBOEBONFNFOEFLLBOEPNBJO)'TBNQBJTFLJUBSEVBQVUBSBOIFMJLBM .PEJmLBTJ )' 4FD" EJCVBU TFJNCBOH TFIJOHHB VKVOH EBSJ IFMJLT UJEBL UFSHBOHHV "LUJWJUBTNVUBO)'4FD"EJVKJEFOHBOLPNQMFNFOUBTJin vivoNFOHHVOBLBONVUBO E. coli ZBOH TFOTJUJG UFSIBEBQ TVIV %BUB LBNJ NFOVOKVLLBO BEBOZB GVOHTJPOBMJUBT QBEB WBSJBO 4FD" EFOHBO )' ZBOH EJQFSQFOEFL TFDBSB TJNFUSJT 4FNFOUBSB JUV TBUVTFUNVUBOEFOHBO)'ZBOHMFCJIQBOKBOHUJEBLNFOVOKVLLBOBEBOZBBLUJWJUBT SecA secara in vivo.VUBO4FD"EFOHBOEPNBJO)'ZBOHMFCJIQBOKBOH 4FD"J EBOZBOHMFCJIQFOEFL 4FD"ϯ EJBOBMJTJTMFCJIMBOKVUTFDBSBin vitro4FMBSBTEFOHBO IBTJM VKJ LPNQMFNFOUBTJ in vivo 4FD"J UJEBL NFOVKVLLBO BLUJWJUBT QSPUFJO QBEB VKJ USBOTMPLBTJ in vitro TFNFOUBSB4FD"ϯNFNJMJLJBLUJWJUBTUSBOTMPLBTJEFOHBOFmTJFOTJ ZBOHMFCJISFOEBIEJCBOEJOHLBOEFOHBO4FD"XJMEUZQF%FOHBONFOHHVOBLBOBOBMJTJT Microscale Thermophoresis .45 LBNJNFOHBNBUJCBIXB4FD"ϯNFOHJLBU4FD:&( EFOHBOBmOJUBTUJOHHJTFNFOUBSB4FD"JNFOHBMBNJLFTBMBIBOEBMBNQFMJQBUBOQSPUFJO (misfolded %BSJEBUBUFSTFCVU EBQBUEJTJNQVMLBOCBIXBQBOKBOHEPNBJO)'4FD" NFNJMJLJQFSBOQFOUJOHBHBS4FD"EBQBUCFSGVOHTJEFOHBOCBJL%BSJIBTJMVKJBLUJWJUBT "51BTF 4FD"ϯNFNFSMVLBOMFCJICBOZBLFOFSHJVOUVLQSPTFTUSBOTMPLBTJ)BTJMUFSTFCVU TFKBMBOEFOHBOBTVNTJ)'CFSUJOEBLTFCBHBJQFOEPSPOHQPMJQFQUJEBNBTVLLFEBMBN QPSJ4FD:&( QSPUFJONFNBTVLJQPSJEFOHBOMFCJIMBNCBUTFIJOHHBNFNCVUVILBOMFCJI CBOZBL"51/BNVO GFOPNFOBJUVKVHBCJTBUFSKBXBCEFOHBOBSHVNFOCBIXB4FD"ϯ LVSBOH FmTJFO EBMBN NFNCVLB QPSJ 4FD:&( TFIJOHHB NFOZFCBCLBO QFOJOHLBUBO QFSNJOUBBOFOFSHJVOUVLQSPTFTUSBOTMPLBTJ0MFILBSFOBJUV IBTJMZBOHEJQFSPMFIUJEBL NFNCFEBLBONFLBOJTNFNPMFLVMFSZBOHUFSKBEJOBNVOMFCJINFOFLBOLBOCBIXB)' CFSQFSBOQFOUJOHCBHJBLUJWJUBT4FD" #FCFSBQB TUVEJ UFMBI NFOVOKVLLBO CBIXB 4FD:&( EBQBU EJMFXBUJ PMFI QPMJQFQUJEB ZBOHUFSLPOKVHBTJEFOHBOTFHNFOOPOQFQUJEB< >4FMBJOJUV USBOTMPLBTJQSPUFJO NBTJICFSMBOHTVOHLFUJLBTVCTUSBUEJMFLBUJEFOHBOEVBnVPSPGPSQBEBQPTJTJ$UFSNJOBM <>%BSJUFNVBOUFSTFCVU EBQBUEJTJNQVMLBOCBIXBQPSJ4FD:&(TBOHBUnFLTJCFMEBO SFMBUJGUJEBLEJTLSJNJOBUJGEBMBNIBMLPNQPTJTJTVCTUSBUOZB1BEBbab 3 LBNJNFNQFMBKBSJ MFCJIMBOKVUQFSNJTJWJUBTTVCTUSBUUSBOTMPLBTJ%BMBNIBMJOJ QSP0NQ"EJHVOBLBOTFCBHBJ TVCTUSBUZBOHLFNVEJBOEJNPEJmLBTJQBEBQPTJTJ/UFSNJOBMEFOHBONFOHLPOKVHBTJLBO nVPSFTFJO NBMFJNJEB 'NBM EBO KVHB QPMJNFS PMJHPFUJMFOHMJLPM 1BEB QFOFMJUJBO JOJ . 108.
(20) Summary. EJHVOBLBOEVBNBDBNQPMJNFSPMJHPFUJMFOHMJLPMEFOHBOLPOUVSZBOHCFSCFEB NBM1&(1000 dan malPEG3000 )BTJMVKJUSBOTMPLBTJNFOVOKVLLBOCBIXBQFMFLBUBO'NBMQBEBQPTJTJ $UFSNJOBMUJEBLNFNCFSJLBOFGFLZBOHTJHOJmLBOUFSIBEBQFmTJFOTJUSBOTMPLBTJQSPUFJO /BNVO Fm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mLBTJQSPUFJOEFOHBOTZBSBUUSBOTMPLBTJCFSIBTJMUFSJOJTJBTJEFOHBO CBJL4VCTUSBUZBOHUFMBIEJNPEJLBTJEBQBUUFSUSBOTMPLBTJEFOHBONFLBOJTNF piggyback. Pada bab 4 LBNJNFNQFMBKBSJEJOBNJLBJOUFSBLTJBOUBSB)'EPNBJOEBSJ4FD"EBOQPSJ 4FD:&(4FDBSBLIVTVT CBCJOJNFNCBIBTUFOUBOHHFSBLBO)'4FD"TBBUUSBOTMPLBTJ QSPUFJOCFSMBOHTVOH6OUVLNFODBQBJUVKVBOQFOFMJUJBO TBUVTFUNVUBO4FD"TJTUFJO UVOHHBMEJCVBULFNVEJBOEJLPOKVHBTJEFOHBOnVPSPGPSEPOPS4FMBJOJUV TBUVTFUNVUBO 4FD: EJCVBU EFOHBO TJTUFJO UVOHHBM UFSMFUBL QBEB BOUBSNVLB 5.C 5. EBO 5. ,FNVEJBO TFSBOHLBJBO NVUBO UFSTFCVU EJLPOKVHBTJ EFOHBO nVPSPGPS BLTFQUPS BHBS EBQBUEJMBLVLBOVKJJOUFSBLTJEFOHBONFUPEF'3&5%PNBJO)'BEBMBIEBFSBIBLUJGEBSJ 4FD"TFIJOHHBNVUBHFOFTJTQBEBEPNBJOUFSTFCVUNVOHLJOTBKBEBQBUNFOHHBOHHV BLUJWJUBTQSPUFJO0MFILBSFOBJUV EJQFSMVLBOVKJBLUJWJUBTNVUBO4FD"ZBOHUFSLPOKVHBTJ EBOUJEBLUFSLPOKVHBTJEFOHBOnVPSPGPSTFCFMVNEJMBLVLBOVKJJOUFSBLTJNFUPEF'3&5 )BTJMVKJUSBOTMPLBTJJOWJUSPNFOVOKVLLBOCBIXBNVUBO4FD",$EBO2$NBTJI NFNJMJLJBLUJWJUBTTFUFMBIQFNVSOJBOEBOQFMBCFMBOEFOHBOnVPSPGPS*"/#%4FNFOUBSB JUV BLUJWJUBTNVUBO4FD"3$ :$EBO"$NFOVSVOTFUFMBIQSPTFTQFNVSOJBO ,POKVHBTJTFMVSVINVUBO4FD"QSPUFJOEFOHBOnVSPSPGPS$ZNFOHIJMBOHLBOBLUJWJUBT USBOTMPLBTJ QSPUFJO #FSCFEB IBMOZB EFOHBO NVUBO 4FD" BEBOZB TJTUFJO UVOHHBM LPOKVHBTJnVPSPGPS$ZNBVQVO5FYBT3FEQBEB4FD:UJEBLNFOHHBOHHVBLUJWJUBTQSPUFJO 4FD:%BSJIBTJMVKJin vitro EJTJNQVMLBOCBIXB4FD",$EBO4FD"2$EFOHBO *"/#%TFSUBTFSBOHLBJBO4FD:5FYBT3FEEBQBUEJHVOBLBOVOUVLVKJ'3&5MFCJIMBOKVU <QFSJNFO'3&5EJMBLVLBOEFOHBODBSBNFOBNCBILBO4FD"*"/#%EFOHBO4FD: 5FYBT3FE4JOZBM'3&5UFSBNBUJLFUJLB4FD"*"/#%EJUBNCBILBOEFOHBO4FD:5FYBT 3FE)BMUFSTFCVUNFOHJOEJLBTJLBOBEBOZBQFOHJLBUBOBOUBSB4FD"EBO4FD:&(4JOZBM '3&5NFOVSVOLFUJLB4FD"ZBOHUJEBLUFSLPOKVHBTJPMFInVPSPGPSEJUBNCBILBOLFEBMBN. 109. 5.
(21) Chapter 5. 4FD:ZBOHUFSLPOKVHBTJEFOHBOnVPSPGPSBLTFQUPS0MFILBSFOBJUV LBNJNFOZJNQVMLBO CBIXBTJOZBM'3&5ZBOHUFSEFUFLTJBEBMBITQFTJm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esimpulan dan Saran 4FD"BEBMBIQSPUFJONPUPSZBOHNFOEPSPOHUSBOTMPLBTJQSPUFJONFMBMVJLPOWFSTJFOFSHJ LJNJBEBMBNHFSBLBOUFSBSBILFEBMBNTBMVSBOUSBOTMPLBTJ<>,BNJNFOFNVLBOCBIXB 4FD:&(NFNJMJLJQFSNJTJWJUBTUJOHHJUFSIBEBQTVCTUSBULBSFOBEBQBUEJMBMVJPMFIQPMJNFS IJESPmMJL ZBOH QBOKBOH (VHVTHVHVT CFTBS ZBOH UFSEBQBU QBEB QSPUFJO TFLSFUPSJ ZBOHEJNPEJmLBTJEBQBUNFMFXBUJTBMVSBOUSBOTMPLBTJEFOHBONFLBOJTNFpiggyback .PEJmLBTJTVCTUSBUQBEBQPTJTJQBEB/UFSNJOBMNFOHBLJCBULBOUFSHBOHHVOZBUSBOTMPLBTJ QSPUFJOTFIJOHHBIBMJOJNFOVOKVLLBOBEBOZBQFSTZBSBUBOLPOGPSNBTJQSPUFJOTFLSFUPSJ QBEBQSPTFTJOJTJBTJUSBOTMPLBTJZBOHNVOHLJOCFSLBJUBOEFOHBOQFNCFOUVLBOBXBM hairpin IFMJLT BOUBSB TJHOBM QFQUJEB EBO EBFSBI /UFSNJOBM QBEB TVCTUSBU EJNBOB NPEJm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nVPSPGPSBLTFQUPSQBEBCBHJBOMBJOEBSJ4FD:. 110.
(22) Summary. DAFTAR PUSTAKA 7FFOFOEBBM",+ WBOEFS%PFT$%SJFTTFO"+. 5IFQSPUFJODPOEVDUJOHDIBOOFM 4FD:&(Biochim. Biophys. Acta - Mol. Cell Res. 1694 o %SJFTTFO"+./PVXFO/ 1SPUFJO5SBOTMPDBUJPO"DSPTTUIF#BDUFSJBM$ZUPQMBTNJD .FNCSBOFAnnu. Rev. Biochem. 77 o #FSH#WBOEFO $MFNPOT8. $PMMJOTPO* .PEJT: )BSUNBOO& )BSSJTPO4$3BQPQPSU 5" 9SBZTUSVDUVSFPGBQSPUFJODPOEVDUJOHDIBOOFMNature 427 o ,VTUFST*%SJFTTFO"+. 4FD" BSFNBSLBCMFOBOPNBDIJOF Cell. Mol. Life Sci. 68, o 7BO%FS8PML+18 %F8JU+(%SJFTTFO" 5IFDBUBMZUJDDZDMFPGUIF Escherichia coli 4FD""51BTFDPNQSJTFTUXPEJTUJODUQSFQSPUFJOUSBOTMPDBUJPOFWFOUTEMBO J. 16 o 4DIJFCFM& %SJFTTFO"+. )BSUM'68JDLOFS8 ϯ)
(23) BOE"51GVODUJPOBUEJGGFSFOU TUFQTPGUIFDBUBMZUJDDZDMFPGQSFQSPUFJOUSBOTMPDBTFCell 64 o 5TVLB[BLJ5/VSFLJ0 5IFNFDIBOJTNPGQSPUFJOFYQPSUFOIBODFNFOUCZUIF4FD%' NFNCSBOFDPNQPOFOUBiophysics (Oxf). 7 o #FDIUMVGU1 /PVXFO/ 5BOT4+%SJFTTFO"+. 4FD#BDIBQFSPOFEFEJDBUFEUP QSPUFJOUSBOTMPDBUJPOMol. Biosyst. 6 o /JTIJZBNB,*5PLVEB) 1SFQBSBUJPOPGBIJHIMZUSBOTMPDBUJPODPNQFUFOUQSP0NQ" 4FD#DPNQMFYProtein Sci. 19 o ,PDI)( )FOHFMBHF5 /FVNBOO)BFGFMJO$ .BD'BSMBOF+ )PGGTDIVMUF), 4DIJN[,- .FDIMFS#.àMMFS. In vitroTUVEJFTXJUIQVSJmFEDPNQPOFOUTSFWFBMTJHOBMSFDPHOJUJPO QBSUJDMF 431 BOE4FD"4FD#BTDPOTUJUVFOUTPGUXPJOEFQFOEFOUQSPUFJOUBSHFUJOHQBUIXBZT of Escherichia coli. Mol. Biol. Cell 10 o )FOOPO48 4PNBO3 ;IV-%BMCFZ3& :JE$"MC0YBGBNJMZPGJOTFSUBTFTJ. Biol. Chem. ;JNNFS+ /BN:3BQPQPSU5" 4USVDUVSFPGBDPNQMFYPGUIF"51BTF4FD"BOEUIF QSPUFJOUSBOTMPDBUJPODIBOOFMNature 455 o 6DIJEB, .PSJ).J[VTIJNB4 4UFQXJTFNPWFNFOUPGQSFQSPUFJOTJOUIFQSPDFTT of translocation across the cytoplasmic membrane of Escherichia coli. J. Biol. Chem. 270, o 4JNPO4. 1FTLJO$40TUFS(' 8IBUESJWFTUIFUSBOTMPDBUJPOPGQSPUFJOT Proc. Natl. Acad. Sci. U. S. A. 89 o "MMFO8+ $PSFZ3" 0BUMFZ1 4FTTJPOT3# 3BEGPSE4& 5VNB3$PMMJOTPO* 5XPXBZ communication between SecY and SecA suggests a Brownian ratchet mechanism for protein USBOTMPDBUJPOElife 5 ,BUP..J[VTIJNB4 5SBOTMPDBUJPOPGDPOKVHBUFEQSFTFDSFUPSZQSPUFJOTQPTTFTTJOH an internal non-peptide domain into everted membrane vesicles in Escherichia coli. J. 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. 111. 5.
(24) Chapter 5. ,FZ[FS+%F %PFT$7BO%FS%SJFTTFO"+. ,JOFUJD"OBMZTJTPGUIF5SBOTMPDBUJPOPG Fluorescent Precursor Proteins into Escherichia coli.FNCSBOF7FTJDMFT 277 o EF,FZ[FS+ WBOEFS%PFT$ 4XBWJOH+%SJFTTFO"+. 5IF':NVUBUJPOPG1SM" UFNQFSTUIFTJHOBMTFRVFODFTVQQSFTTPSQIFOPUZQFCZSFEVDJOHUIF4FD"CJOEJOHBGmOJUZ FEBS Lett. 510 o 5TJSJHPUBLJ " $IBU[J ,& ,PVLBLJ . %F (FZUFS + 1PSUBMJPV "( 0SGBOPVEBLJ ( 4BSEJT .' 5SFMMF .# +SHFOTFO 5+% ,BSBNBOPV 4 &DPOPNPV " -POH-JWFE 'PMEJOH *OUFSNFEJBUFT1SFEPNJOBUFUIF5BSHFUJOH$PNQFUFOU4FDSFUPNFStructure 26 oF 5PNLJFXJD[% /PVXFO/%SJFTTFO"+. 1VTIJOH QVMMJOHBOEUSBQQJOHo.PEFTPG NPUPSQSPUFJOTVQQPSUFEQSPUFJOUSBOTMPDBUJPOFEBS Lett. 581 o. 112.
(25) Summary. 5. 113.
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