TheBiophysicalSociety of Japan General IncorporatedAssociation vsuali7ed bv elLctren miLrescop} But the "hole structure ot thL needle Lem plex has not been shown ya In this studv we shew the whole stmcturc of thc needle complex on the cell surtace and describe thc differenLe and resem blance between P aLrugmesa and S typhimunum T3SS 3P243311235 Kazunori Kawasakil Klluht[oA))ama4 h-M)xrv7U-J( 770fip-V7Vhe[SeNa'fK' ATPage opRptN-coanas Intramembrane part]c]es of Na'IK' ATPase blr freeze fracture

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... ephca made with carbon Tum( kt Kttnl Lnuko KDbnyahhil Mtsh Lvv rahlr" YuLaro H-ash l (IAISI 2k)ertnltniversTtv IRiken 4JLIJapan) Freeze fracture rcpl]ca is 'i useful method Lo xisualize membranL pioteins as mtramembrane pdrt]c]es (IMPs) in the conventlonal rephLa method fiactured hydrophobie faLeb of membranes aiL Leated with platmum (Pt) backed with earbon (C) dnd thcn the Pt/C films are uged fbr TEM observation after removng the biologieal materials The elcction densJty ot Pt enablLs high contrast imdging ef template shape of membrane piotems For exam ple Na-IK' Al/Pase Ldn be vTsuahzed ab distinLt IMPs each ot "hiLh hds a rmg like shape Rather than the outlines ot the "ng hke iMPs the helcs m Lhe rmg hke IMPs s a mattcr oi more mterest becaug"he "rcumferences ot tht holes may directly contoui the bhape ot the MPage Smce tlte granulamty of eiaporated C is smaller than that of evaporated Pt theresoluLionoftheIMPimagegmaybeimprovedhyusmgCreplicagingtcad of PtfC replicas Here we hdie tegted thc feasibil]ty of C replicas lor the gtud} ot Na+/K+ MPase The memblanes Lontdinmg thL preLem "ere free7e trdctuied dnd rotarL shadowed b) C dt an angle oi 45J dnd vLith a th]Lkness of 25 nm at 144MC {Balzers BAF400D) The samples -cre observeded by ThM (Philips CM200UT 200kV) and by STFM (FEI Tecnai F20 200kV) Images of -ng hke IMPs were aLtually obtained from the C rep]icas i c sinall heles ln thL IMPts uere obviously dlseerned Analys]s ot the imdges ot the holes m the IMPs on C rLphLas and on Pt/C rephLas will contnbute to thL undcistandmgs ef the oligomei struaure of the MPagc thc asvmmetriL unit It was shewn that the Laroteiioid bactcnoruberm bmds to crevices bLtwLen aqJacent plotem subunits ]n thL tmmeric assembly [2] Besides seven uans membrane helices (A to G) that charelctense archaeal rhodopsmb the phR protomLr potssesses an amphipathic fv hellx (A ) at the N terminus This hdix togetheT wlth aleng loop betwccn helices B and C forms dh}drophobiccapthatLoverstheext]acelluldrsurfaceandpreventgarapidion exLhdnge bet"een thL aLt-e centre and the extracettulal medium The retlnal bound to L} s2S6 m hetix G takes on an alt trang conhgurauon and thc SLhiff base interacts "ith Asp2S2 and a chlonde ion the Iatter being fixedby two po lar gioupb in helix C Tn the amon uptake path"a} four ionisable residues and sei en water moleculcs are altgned to term a long hydregen bondtng network (onversely the cytoplasmiL hilf is fi]led mostJy by hydrophobic residueg foimmg a large energLtiL belmer against the trdnsport of amen We have becn pLrformmg structural analyges of pHR in complex with anothcr type of amon (nitrate bTomlde or amde) Refi.rLncets rll K Ihara LL al {200g) FEBSLLtt 582 2931 2936 [21 T Kou>ama etal QOIO)J Mol Biol 396 )64 579 3P246 INafUtM 3P244311250 Sou"KHn"du vetstt)) NatronornonaspharaenJsM"opveMitfi$trl)irt;,7 /N P"FV')),diOrpnvtsdiStettueopva.H.msva Crysta] structure of an O ]ike blue form of the Iight driven chloride purnp halorhodopsin from Natronomenas pharao mslsutomuKouyma CDcparimenrofPh}stcs firftdTtatE SchoolofScience Vhgo}a Vnt The 1ight driven chlonde pump hdlorhodopsin (pHR) isotated trom the hatorhodepgin oberpioducmg gtrain KM 1 of ptatronomonas phardoms "as crybtalhzed tnto a rnonochniL C2 Lrlr stal -ith a pHR tmmer in the as} rnmLtnL un]t Tht previous cr} stailographiL sLud} of the chlondc bound purple form ot pHR showed that thL reunal Schiff base intLracts with Asp252 and a Lhleride ion thc latter bemg fixed by two polar groups (Thr126 and Serl3e) in helix C [1] In this study we carnLd out structural analyses of ali anion free O hkc torm of pHR This blue torm wag genLrdted m a post LrysLdl]idtJon soaking solution m which only bulky anionts (sulphate and gl}ulle) were contained The structural anal}gis showed that the retina] beund to Glu256 takLb an all tr'ms configuratlon -ith the Schiff base bcmg hlrdrogen bonded Lo Serl30 in he]]x C The most remarkable diiiuenLe bet-een thL Lhle-de bound purplc foirn dlld the amon free blue form was obscned m the chtoridc uptake path way 1 e upon the remova] ot anlon a fiip motion ofthe sidechain of Glu234 wats dccompanied by 1argc mevementg of thL surrounding aromatk retsidues (lyr124 Trp127 Trp229 and fyr248) Conversety no 1arge movements wLrc obberved for the other intcrndl iomsible residuLs (HislOO Nrg123 Arg176 and Asp2S2) diid the gtructurc et the L}toplagmic halt was scarcely affccLLd b; the anien rcmoval ReterenLe 1)T Kou}ama S Kdnada Y Takeguthi A Naiusawa M Mu rakaim dndK Ihara C2010)J Mol Biol 396 564 )79 NatronomonaspharaonisMXopNpm#Mfi.r7t)trf)i7.A m m F 7S, ).O X tuza .-.asfineut X ray Crysta]]ographic stlldies of the Light Dmven Chlomde Pump Ha]orhodopsm ftom Natronemonas pharaoms Soun Kar )du1 Jm Znngl Yu TlakeguLh" Mdden Murakelmii Kunio Ihara' eradstax. VthottlofScFente Nago)aL uieM b ICent,rforGene Rt tEarch Chlkita ki Vcgcna464 S602 Japan) Bacteriorhodepsm is a menibrane protein that funcnons as a 1ight driven pro ton pump Previous Lr}stallographic studies ef the hexagonal P622 crystal ot -i1d type bacteriorhodopsin (bR) have ghown Lhat rotdtion ot the side chain ot Leu93 asgists waLer relocation aLross the retsnat Schiif base m the K to L traii sitlon So it is interesting Lo irrvestigate hew this water reloLation is affected by replacement of Leu9a by a gmallLr ammo acid rLsidue Ala Further, m the photec>cle ofbR mutant L93A high conLentrations ot an O 1ike mtermedlate aceunluldte This effeLt has been atributed Lo an inhibitcd reisomerization et its 13 cis retinal ehremophore to thL all trans foTm -hich causeg an abeut 200 fold increase ot the O deca} ume So the structurL of the O mteremdiale which has not yet been eluctdatcd can be expectcd m thig L93A muLant In this btud) we crystalhzcd the Leu93 te Ala muttnt baaemorhodopsin mto 1 hexagonal P622 crysta] with eell dimensiong ot a = b!: 102 2A and c = 1 12 2 A Diffraction data al 2 2 A resoluuon showed that the Leu93 to Ala replacement eaused no large alteraUon m the oven]1 stsucture of bR and that the cavity created by thc replacement are occupied by three additional watcr molecules The mtermediateg ot bR L93A mutant is also m pTogress 3P247 ibkaaki Idhika-a; (] l)epr oj hnknal Yt T O fingtnEennA jorLfarerfrlsResearch 7SlasvexthnLkmarrM6"CSmprttrJVma-fiC[es;t6.rl ytage*$ft7EJv77xyu=ywt lntcrfh"al ion conductiv]ty betwcon audic or ne"tral gels contain]ng ammo ac]d, and hydrogenated amorphous sihcon film TakL}a hkdhash" Htreshi Misumoto2 ThkEsh eeto' YLiraka Ts-iuLhii lkita Lmt 2Cenrer fitr JntmdTrctph ian Reseajch XJnstxrtttc 7bttokuln!}) 3P245311305 Tsntomu KDuva]na1 {IDepartmensofPh)slcs NagovaLts-ersm The hght d-ven chloTide pump halorhodopsm (pHR> of Natroiiomonas phdraonis was isolated from thL halorhodopsin (hR) overprodueing strain KM 1 [11 The membranc fusLon method wag uUhLed to crystaIhLe pHR into d monochmc crystal belongmg to the space greup C2 wlth a pHR tnmer m Wtl hafoe been inNesugated in the ploblem ho" to design an arttheial bio nioleLulai gel film foT LonstiuLtmg a bio molLLular devicL that hds a highly Lontrollabte interfacial ion conduLUve property onLo orgamsrns Tb provide an effectlve way to constiuLt an elrtificial bio hlm that can be safely attached to a livmg body or an orgdmsm and Lan transport ion mto them reLently we have bcen designed a gel gel interfacial ion conduction gystem ugmg ch fferent two amino aeld as additiieg ln this study intertaual ion conductivity bet"een acldlc or neutrdl gels contdming amlno acid bTistl} glutamic acld(Glu) as partic aeidC4tsp) gtutammL(Glll) proline(Pro) dnd gl}cine(Gl}) were usLd ds additiies to agarese gel Gel gel mterfaciahon conduction was analyzed by voltelge currLnt measuremcnt Several patterns Glu Gl} Glu GIn Glll Pro Agp Gly Asp Gln oi Atsp Pro of gd gel interfhcial ion conduetion were Lompared Seeondly a hydrogellated amorphoub sihcon (a Si H) fi1rn on trangparenL conductive hlm "as attaehed to the ge] gd mterface ThL$ was Londucted because that Lontrolhng the electnc fidd weakty by usmg a semi Lonductor hlm prepared "tth tugh preciston is eflLLtive to ion transport on the film surface Light mduLed gel gel mterfacial ion Loiiduction change -as anal)zed 3P2487xlristzueafiblve7Jv\=y=nblvopzzwhc[estt6 (ttztagcogetatetreasiHoinm Rcctificat]onpropertyofmterfacia]]onconduct]vitybctween gelb contain]ng aspartic acid or argmtne, and effbet ot a Si H -Sl88-NII-Electronic