TheBiophysical Society of Japan General IncorporatedAssociation 2P189 ATP-driven rotation of VoVl from Thermus thermophilus C)MasahiroNakane',MasatadaTamakushi2,HiromiIrnamura',MasuHukeYl)shidui, KenYbkoyaima" 'Tokyo [nstitute of Technn]ogy, ]Tokyo Univcrsity of Pharmacy and Ltt'e Science, !OsakaUniversity.11apanScienceandTcchnologyAgency Xlacuelar-typc H'-ArPases (Vf\IrPases, XbVl), which are coinmonly found in most eukaryetic cells. function as a proton pump with consumpticm {)r MP. The

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... gs of eukaryotic VMPase are also found in seme bacteria. such as a inermephilic bacLerium. Ikei'mus thermopltilus. We previnusly deinonstra:cd thc rotation el' beth the DF reter ill Vl and the su)-ring in V-Al/Puse drh'eii by AI/I' hy{irolysis, Here, we report seme progresses of obsen.ation systein for the rotation of holo V-Al'Pase with At'P hydToTysis. We isolatcd thc Nlo with histidinc tag in thc M]-ring from T thernzophilus membrumes. The Avj-tag sequence was introduced into the each A subunit in the V1 t'rum cNprcssion system using E. coli. The mutunt holo VL rtrPuse was recenstituted from the isolated V1 and M]. By using thc rcconstituted V-NrPase, the frequency of rotating molecules was drastieally increased, Then, we obsen'ed stepwise retation at low an'P concenrrations. The rotztional speed was almost propsrdomal te MP concentratiuns between 10 and SO psM, indicuting that binding of rtI]P to the NbVl is a rate-limiting step undcr thcse conditions, By l'itting the 1 'stegrums ef dwell times with a sillgle exl}enential cquation, k." fer irrP was estimat:d te bc 5.3X 104 M".s", xvell coisistent with bulk phase data. Under the same conditien, the eslimated k.. of V1 was 1 .7 X 1Oi M'i.'i, whlch is about 3 times hig]]cr thuri that of VMPase, This result indicates ChaL binding of Nb Lo V1 domain uffects catalytic properzies in Vl domain. 2P190A novel expression system for dominant negative mutant actins OTuro Q.P nnguchi"'2. Hironori Ucnoi, Kctko Hirosei, Taro Q,PUyeda' iT.ife und Environmental Sciences. Univ. of Tsukuba, "Research Institute for Cell Engineering.AIST It is gcnerally acccptcd thaL myosin gencrates force by nlting the neck domuin. Hc]wever, several studies suggcstcd that rnyosin binding induces cooperative cenforrnutional chunges withiii ttctin filaments, which play important rolcs in myosin movement. For instance, An et al {l996) identMed dc)minEtnL negutiye (DN) mutant actin allelcs that irnpair fiight of D, melanogastor eyen in tiie presence ef three-fold excess norma[ actin gencs. Thcsc phenot,ypcs arc censistenL with the idea LhaL the DN uctins preyent presumptiye myosin-jnduced coopcrative cunformati"nal changes in autin filuJ]ents, but biochemical analyses have been hampered by difficulty in purifYing DN actins frem fiies. Expression of rccombinant actin has also met difiiculties because direct epitope taggifig tends to uggregate actin. and furthermore, DN aetins are cxpccted te bc toxic for thc host cells, Thereiure. using Dictyostelium cejls, we expressed and pllrified aczin that was fused C-teiniinally to -is-taggcd thymc sln betu 4, an uutin menemer binding and sequestering protein, and rcrcascd intact actin by eleavage with chyrnotrypsin imrnediate]y downstrcam of thc mativc tcnninal Phe residue. Wild-type recombina]z actin obtained thjs way polymerized norniatly as exarnined by sedimentation assay and elecLron microscc)py, and moved at neurly normal speeds on surfaces coated with skcletal HMM. Wb arc cxprcssing e[tch DN aetin using this nevel express'en systen]. We found that G63D DN actin is unablc to polymcriie, and are imvestigatin.v why this exerts deminanlt ncgativc et'fecr. 2P191 Mechanism of' mechanical work and efficiency enhancement in myosin filament assembly C)Motoshi Kaya. Hideo Higuchi BiomedicalResearchOrganization.Tol}okuUniv Recent opticul trapping studies of individual motor proteins have cnabled us to guiii insight illte the mechanism of muscle contraction. It Ls welL known that myosin II is nen-proces$iye, and so, myeslns havc to asscmble into a thick filament to produce force continuousty as required in muscle. Thus, it is very intriguing te ask the question/ How do individual myosins assernbEe inte a thick filament to become a i'orce generator wiLh the maximum efficiency as observed in muscle? Therefore, we investi.cra[ed displaccmcnts (d) and 1'urces CF) preduced by different lengths (molecule numbers) of skeletal muscle myosin fitaments uslng the optieal trapping na"ornetry. Both shert (O.7urn) and Song (1.2um) filaments processjvely produccd discrete stepwise mevements uveruging 5-6nm imd produced the 6 and 12pN peak forcc, rcspcctively, Thc stepping efficiency (e: Lhe raLie of forward sLeps to baukwurci steps) vvas 1iiglier for long filaments thall short filaments at any given load. Thus. Ihe velocity and inechanicul werk {FXdXu') per filament lengtlt produced by long iilamenLs were higher at any given loud, The seeond-order rute consiant for asP binding calculatcd by using the mean dwell time was similar te the value for the acto-myosin AI/Pase. Ihus, each step presvmably corrcsponds te une rtrPasc cycle. Frem these results, we concluded that the increase in myosin molecules enhances the degree of unidirectional movement, resutting Lhe increase inthermodynamiccrnciency(mechanicalworkrfreeenergyofM/I'hydrolysis), ComTnunications betwccn the mack-binding sitc and AI/Pase site are essential fer the mutile actiyiLy er motor pioteins, In dynein, its MPase site(s) is located within the head domaifi containing six AAA+ medules, whilo Lhc micT{)tuhule {MT)binding site called stalk hcad is spatLally separated from the head domain by a le-15 nrn slender stalk that js predictod to bc formcd fivm an antiparallel coiled ceil (CC). Therefore, the lirformntion at the NI]Pase site and MPbinding site of dynein must bc traiismltted throvgh thc long stalk CC. Ho-veveg Lhe mechanism unde:lyingthisbidirectionalcoinmunicationsreniainsunclcar Recenlly, it has heen proposed thar shifts in thc a]igumcnt of two hcliees forning the stutk CC rnodutate the MTLbinding affinity of the stalk hcad (Gibbons ct at. <200S)), Hcrc wc employod a disull'idc cioss-linking strutegy to eluciduto the reles ef the predicted shifts within thc stalk CC in thc bidircctienal eommunications. Our results showed that when the two helices were eross-liirked by a disulfide bond, the dynein displayed MTindependent "activated i' NrPase activity and trapped tn an AI]P-insensitii,e iistrong-binding " sLate. Cunversely, ufter unother type of crosslinkuig that is cxpected to causc a $mal1 shift in thc alignmcnt ot' lhe CC, the dyneii exhibited vaLindependent "basal'i tVPasc aetivlty with an MP-inscnsitive "weak-binding" state, 11hese rcsults strcmgly suggest that dynein utilizes smal1