of Mn stts eaktshuln [buw-Tny) of theCI)eparr tdg dthnity chromatography Human tubutin thus punfied was polymenzed mto filaiiients in the prebenLe of GTP and taxel The motility ef kmesin was conhrmcd m the microtubule g]idmg assay Thc results of fuTthLr efforts on both humdn and Drosophila tubutin -i11 bL iepoited dt the meeLmg Fl MPase s a retary moletuldr motor m whiLh a central 7 subumt rotateg agamst hexagonally arrangcd subumtg {iifi3 hydrolyzmg MP sequentially m three fi subumts It

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... challengmg to uncoNcr coniormational ehanges ofthe Fl complex dumng iotakon We measured the conformdtional changes ot the Fi complex using two methods First attaLhing a Targe pTobe (40-・500 nm in diametei) to the ), gubunit results in amplifiLauon of movement ol the v subumt thug allowmg us to detect gmall Lonformational changes of the Fi complex MLasurmg displacement of Lhe probe attach]ng FlC6E190D) with el few nanomcter resolution we tound a LirLumradius ot the catatytic d"ells was 2-・4 nm larger than that of the MP waiting d-ells This rLsult fuiors a tiltmg{-40)ofthe7subunitinsldLa]P]dtthetransitlonfromtheMPwartmg dwell to the catalyuc dwell Secondly we apphed single moleculL rRETmea surement to detect distdnLe chariges between the fi subunits m Fl oslt190D) havmg d FRhT pair {Cy3 and Cy5) We tound the transitien bLLween high (-e g) and low (-・O 5) FRET eMciencies at high MP concentadtions (abefoe 1 "M) tn which AI/P "amng ti]ne"as neghgible at the ume resolution (1O-30 tramtlsec) Considenng crystal structuTes low FRET elhcienc} mdicates one I3 subumt m the opell form but another m the Ltosed torm On the other hand high FRET eficiency mdiLates both ot the6 subuntts tn the closed form We are pcrlormmg further expenrnents FoFlfyr?antptficoIenmevyym% gKlfievts6bo :s&coreregftont ± NonResonanLe Raman Eyidence for Conformat]onal Changes m the Fo Subumts by Substrate Bmd]ng m Boyme boFl iYI/P gynthase M]noru Kubo' Ksoko Sh/]Lel-a Irohl Tikaghi Ogu[aP Shinlra Yosh ltail 1 Sfienw Gmduart S[hoot ofLrfe Sr"ncL UnxLertit) of H>oso 2PtcobmtogT bmTeisityofHiogo) 2P204 1lakashiKouda rtates[tmotojeng!ne,1tng "Jvi)ivyeurfv-Aexureukca2+rcumaagfienms emueasee7"eilvFJvcowafi Deyelopment of molecular ghuttle that has a calcium depen de]it cargo load)ng svstem us]ng calmodu]m and iiposome Hideki ShiEiudo Iroshi Takihatd ShinMaku MaTuta COiviston ot bioipt"7nar"s Grnd SekaLn"erstv) 2P202 Kaoru Mtedal clDepartenettt oj Lofe lnsnt"ge arad"atL 5citootofLije Sewnte F.Fi MTP synthase is a large protem complex <550 kl)a) that cataly7Lg MP synthasis dnven by a transmembrane proton gradient Proton tlows through thc membrdne intnnyc Fo part whereas rtI/P is bynthesized in thL membrane superficial Fl Although MP s} nthLsisthydrelysis reaetion has been gtudied ]n detelil based on eTystal btructures ofthc ]gelated Fl the couplmg mechansm between proton transloLatien and ATP synthetsis remains unknown due to thc lack of the structural analysis of the F. at the atomiL level ln this btudy we report non resonancL Raman spectra for FoFI MP synthase fiom bovme healt mitochondna in the absence and piesence of AMP PNP a iion h}drolyzable ATP competttive inhibitor The pumfied enzyme Lontained ail the 18 subumts m both the F. and Fi so that Raman speLindl changes by addition of AMP PNP wats attnbutable to contennduonal change m the who]e en7yme Notable TYr doublet Raman bands dt 83S and 852 cm'i suggcstcd weakenirrg ot the hydrogen bonds ofbome TYr -heieds d Trp Raman band dt 882 cm d Teiealcd that seieral Trp experience merL hydrophobic envirollment This ent} me Lon tains one Trp in thc E subumt of Fl (at the mterface with the F.) and l8 Trp in thL stator subunits of Fo Thus the pregent Raman result dLmentstrateg that substrdte binding to the Fi mduces a glebal Loiiformauonal chaiige includmg m the gpatLally remote F. gubunits 2P203 EtamavaeMLIte+z-7VY ± RreMkOcait Deyelopment ef a high y]eld tubuhn expression gystem in baculovirus mgect cells Ybll Hnch]kubu Se"chl UctnmLra Etsuko Muto (Bnnn Yt"ncebtsrtxute RtifEN) Formutational andiysis oftubulm wepieviougl} deieloped an expressioll gys tem for tubulin uging yLagt Lelts and succeeded in identltymg the key residuLs invo]vedmkmesinmotihty(Uchimuraetal 2006 201e) Hew"ei thatyeast expression sygtern is hmited in its application because of its low ylcld trom 6 hLerb of yeast culture we usual]y obtam a maximum ot 500 ug ot tubulm In order to use the expresged tubulm m structural and biochemical andlyses it is necessary to obtain a largci amount of tubulm preferably moie than IO mg from a single preparation Aimmg to develop a high yield tubulin expression system we dttempted to Lxpress and punfy human and Drosophila tubulm usmg baculovirus insect cells Using baculovirug insect eells we have so far suLceeded in exprcssmg dnd purltymg SOO tig ot human tubu]in flom d 1 titer culture (= ]e g ofcells) -ith a pu-t} higher than 90% To gcpardte human tubuhn frem the endogenous tubulin in lnscct Lells each cr tabuhn (TUBAIB) aiid fi tubul]n (TUBB) wag fused to His and FLAG tags at the C terminug respectively dlld human tubulm dtmer was purined through suLLessivc runs ef cell extracts in anLon exchange eolumn chromatogidphy and His and )LAG RLLently attentaon ig hemg focused on thL applicatron o[ moleLular shuttle based on kmeyn ind rmcrotubules kssential pomt regarding a moleLuldr shut tle is the cargo ]oading system Sinec the mt-nsic physiologiLel1 mechanism underlymg the selective bmdmg of Largo to thL tail domain ot krnesm hels not beeii Ltdrihed thus far athficial binding sybtems haie bcen uti1ized tor cargo bindinbg Foi mstance in a prLvious study blotm avidin and anugLn antibody systems were uscd tor Ldrgo binding These systerng are known to be highly specific with extremely strong bmding property However they have Lhe draw back that thc shuttle cannot releasL the cargoes Thuefore it is required that a moleLular shutt]e should have the property of reNersib]e bmdmg of cargo In this study we developed a new mo]ecular shuttle system in which the car goes of hposomc labeled with M13 peptide reversibly bound to the shuttle ot kmesin CaM fusion protein dependmg on Ca2' concentration We have preparcd the tshuttle K560 CaM that consists of the N termmal motor do main neek region a helical eoiled coil region and CaM dt the C termmus KS60 CabM showed Ca2' dependent rLveisible bmditig Lo M13 GFP And Maleimide Longugated 1ipogome wels prepared aad its stabihty as a Largo wag exdmined Wc alse designed and gynthesized the M t 3 peptide that hag a smgle cygtun at N terminus The peptide was reacted with ma]eimlde located on the surfaee of 1ipogemL Currentl} we ai L studymg the Ca2+ dependent inIeraLtion betweenM13 hposime andM)60 CaM 2P205 Actm bundlmg prutem IQGAP regulates distnbution and stabi]ity of myosm II ]n the contract]lc rmg m fission yeabt Masak Tlakume Osaniu Numltd Kcntaro tsfikm/o CDepartment oj StruchfralBiogcienteL Graduate Sthottl ofrde andEnutenrnental Sclences Ln/-ersih (v'Tfiukuba l J l nnntthdat lstakub[x JbarakT SOj Sj77 Japctn} Fission yeagt Sfhi7osaccharom}ces pombe undergoes bmary figgion durmg Lytokinesls usmg the actomyosm Lentsactile nng (CR) 1ike anlmal Letls An aLun bundlmg protun IQGAP Rng2 its mvolved m the generation Df CR F dcun and Lheir arrangement mto a ring (Tlakame et al (2009) EMBO J 28 3117 31) Here we report that Rng2 is implicated also m localization of myosin II (Myo2) m the CR ln eaily mitosis Myo2 dccumulatL as d band of fine dots en"rLlmg thL cell eguator in an F aLtin independent mdnner and then Londenses lnto a rmg via lnteracLien with CR F dLtin The punetate to calization ef Mye2 was abolished in a temperature sensitive mutant of Rng2 Morwvei FRAP anatysis revea]ed that Myo2 m the CR turned osei more rapidly m the mutant cells than in wild type cells ThLbe results may suggest that Rng2 reguleltes distmbution dnd gtability ef Myo2 in the CR by at lcast two distinct mcchamsms to ensure succegsfu1 cytokmesig 2P206 ?fima:-IYtz I C[*5 F77f),Mdtopfiemeq-Regulatory factors for F actm bundhng mduced by smooth mllsclemyosmI Yoh OkamDto Koti Nobuyuki imhei Shihiki Titsuya Kav-ai CDivivon ofAmphLd Sctence andEngi neertns lfuroran Jn"ntme oj Zchnatog;) Actm ftlaments are occasionally crosg I]nked and bund]ed by actin bindmg proteins in cellb It hag been ieported a model for smoeth muscTe contraL t]on includmg the assLmbly ofcyteskeletdl and extraLettulaT matllx eldhesion complex protem at thL membrane Thc protein cemplLx mduced thc po]ymer i"taon and orgamzeltion of membTaneous network of actin fildment In fact aLtin Lytoskeleten m vascular smooth muscle has been known to be remodLled dunngtheLontrdction PorLme dertel m}osin I is a singlc head motor pretem with calmodulm bound to the 1ipid bmdL]g short tai] We haNe tound a myoynI Lnduced F actm bundling at physiological iomc condiuons Furthcrmere the F aetm bundlmg hag bcen inhibited by phosphattdyl glycerol but not with phos phaudy] chohne(TKawai etaE2009) These obgervalJons werc collsistent with om preiioug result of the bindmg specificlty ofphogphohpid to myosm I (Hasegd" a Yet al 1996) Both of the head and tdit region of m} osin I nnght be essent]al ior F actin hundlmg through Lrosshnk]ng different strands of F aLUn rcspectiiely lt has also been observed that IP3 and lor PIP2 pToduced strong inhibitory Lffec.ts on thL inyosin I dLpcndent F actm bundlmg with charaLtLr -Sl18-NII-Electronic