www.sciencemag.org/cgi/content/full/316/5821/120/DC1

akiba-online  时间:2021-03-20  阅读:()
SupportingOnlineMaterialforAnATPGateControlsTubulinBindingbytheTetheredHeadofKinesin-1MariaC.
Alonso,DouglasR.
Drummond,SusanKain,JuliaHoeng,LindaAmos,RobertA.
Cross1**Towhomcorrespondenceshouldbeaddressed.
E-mail:r.
cross@mcri.
ac.
ukPublished6April2007,Science316,120(2007)DOI:10.
1126/science.
1136985ThisPDFfileincludes:MaterialsandMethodsSOMTextFigs.
S1toS4ReferencesPageS1SSupplementaryMaterial|Alonsoetal|ATP-GatingmechanismofKinesinSupplementaryMethods1.
Proteins&proteinchemicalmethods1.
1Proteins|Nkin460GSTcontainingtheN-terminal460aminoacidsofNeurosporakinesin-1andaC-terminalGSTfusionwaspreparedasdescribed(1).
Rat430GSTprotein(2)usedinthesteadystateATPaseassayscontainedtheequivalent430N-terminalaminoacidsofratKinesin-1andaC-terminalGSTfusion.
ThisproteinwasagiftfromDrIsabelleCrevel.
RatrK430asusedintheFPLCgelfiltrationandthekineticsexperimentswasuntagged.
TheinsertforthisconstructwasPCRamplifiedfromafulllengthratkinesin(kif5cclone)originallygiventousbyScottBrady.
StartingOligo:5'-CCGCTCTACATATGGCGGACCCAGCCGAATGCAGC-3'EndingOligo:5'-CCGACTAGTCTAGAATTCCTTGTCATCCAGCTG-3'.
ThePCRproductwasdigestedwithNdeI/SpeIandligatedintopET17b.
Expressionandpurificationoftheproteinwasaccordingto(3).
PigbraintubulinwaspreparedaccordingtoMitchisonandKirschner(4)(http://mitchison.
med.
harvard.
edu/protocols.
htm)andS.
pombetubulinbyamodificationofthemethodofDavisetal(5)tobedescribedindetailelsewhere.
PurifiedtubulinsweredesaltedintoPEMcontaining20μMGDPbeforestorageinliquidnitrogen.
ProteinconcentrationsweredeterminedbyUVabsorptionscanofsamplesofproteindissolvedin6Mguanidinehydrochloride,correctingforbackgroundscatteringbysubtractingascanofbufferalone,andassumingfullnucleotideoccupancy.
1.
2SteadystateATPase|ThemicrotubuleortubulinheterodimerstimulatedATPaseactivityofkinesinwasmeasuredat25Cusingapyruvatekinase/lactatedehydrogenaselinkedassayasdescribed(6),exceptthatmicrotubuleswereassembledinPEM(7)containing1mMGMPCPP,pelletedandresuspendedinPEMcontaining10nMGMPCPPbeforedilutionintothelinkedassaybuffer(20mMPIPES,pH6.
9,5mMMgCl2,1mMDTT,0.
1mgml-1BSA).
TubulinheterodimersweredilutedfromastockinPEMcontaining20μMGDP.
NAD/HabsorptionmeasurementsweremadeinaCary50spectrophotometerusingdisposable50μlUVtransparentcuvettes(Eppendorf).
ValuesforVmaxandKmwereobtainedbyleastsquaresfittingtoplotsofATPaseversustubulinheterodimerconcentration,usingKaleidagraph3.
6.
4(SynergySoftware).
ATPasevaluesareperkinesinhead.
1.
3Superose12gelfiltration|Stockkinesin(typically69Mheadsconcentrationin20%glycerol,20mMphosphatebufferpH7.
0,2mMMgCl2,0.
3MNaCl,0.
1mMDTT)wasmixedwithstocktubulin(typically58μMheterodimersin50mMPIPESpH6.
9,0.
2mMMgCl2,1mMEGTA,0.
1μMNa-GTP,30%glycerol)andadjustedtoafinalvolumeof240landfinalconcentrationsof6.
5μMkinesinheadsand13μMtubulinheterodimers,witheithernoaddednucleotide,or2mMAMPPNPor2mMADPbyadding10xcolumnbufferandwaterasnecessary.
Themixturewasincubatedfor10minsoniceandcentrifugedbrieflyinamicrofuge(thiswasprecautionary:nopelletwasobtained).
Theentire240μlsamplewasthenappliedtoaSuperoseHR10/300GLcolumnheldat4Candrunningat0.
5mlmin-1.
Thecolumnbufferwas50mMPIPESpH6.
9,2mMMgCl2,1mMEGTA;pluseithernothingor2mMADPor0.
2mMAMPPNP.
0.
3mlfractionswerecollected.
ThereducedconcentrationofAMPPNPinthecolumnbufferwasfoundtobeadequateandwasusedforreasonsofcost.
Elutionpatternsweremonitoredat290nmtoavoidthelargenucleotidecontributiontothe280nmabsorbance.
ThekinesinabsorbanceislowbecausetherearefewtryptophansintherK430sequence.
Thisisconvenientbecauseitmeansthatthetubulinbehaviourdominatestheelutionprofilesfromthemixtures.
PageS2S1.
4Gelelectrophoresisanddensitometry|SamplesfromthepeakfractionswererunonInvitrogenNuPAGE10%bis-trisprecast15wellgelsandruninMOPS(Invitrogen)bufferat200Vfor1.
5hours.
GelswerestainedwithSimplyBlue(Invitrogen)andimagedwithagreyscaleCCDcamera.
QuantitationwasinitiallydoneusingImageJ(Rasband,W.
S.
(1997-2006);http://rsb.
info.
nih.
gov/ij).
Integratedopticaldensityvaluesforeachbandwerecorrectedbysubtractingtheintegratedopticaldensityofacorrespondinglocalareaoffeaturelessgel.
Thesystemwascalibratedusinganopticaldensitywedge.
Inlaterwork,weusedthesyproorgangefluorescentstainingsystem(MolecularProbes,methodexactlyaspermanufacturer'sinstructions)incombinationwithaPhosphorimager(MolecuarDynamics)andBioRadImageQuantsoftware.
Thetwoapproachesgavecomparableresults.
ThenumbersquotedinthetextreferonlytothePhosphorimager/Syproreddata.
1.
5MantATPturnoverexperiments|Stocktubulin(seeabove)wasdialysedfor30minutesagainstalargeexcessvolumeof50mMPIPESpH6.
9,0.
2mMMgCl2,1mMEGTA,10%glyceroltoremoveunboundguanidinenucleotides.
TheconcentrationofthedialysedproteinwasdeterminedbyUVabsorptionscan(seeabove).
Enzymereactionsweredonein20mMPIPESpH6.
9,2mMMgCl2,usingquartzfluorimetercuvettes.
FluorescencetransientswererecordedwithaCaryEclipsefluorimeter.
Atthestartofthereaction,cuvettescontained1μMMantATPin20mMPIPESpH6.
9,2mMMgCl2.
Tothisweaddedkinesintoafinalconcentrationof1μMkinesinheads,andinsomeexperimentstubulintoafinalconcentrationof2μMtubulinheterodimers.
Thenwe"chased"byaddingeither100mMNa-ATPor100mMAMPPNP,to1mMfinalconcentration.
Thefinalvolumeineverycasewas600μl.
Experimentsinwhich4Moftubulinwasaddedyieldedessentiallyidenticalresults(notshown).
PageS3S2.
SupplementaryData.
2.
1Superose12gelfiltrationofkinesin-tubulinmixturesFigureS1|Superose12chromatographyofkinesin-tubulinmixturesThedatawereobtainedasdescribedaboveandforFig.
2inthemaintext,andtheaxesareequivalent.
Thedifferenceinthiscaseisthatamixingratioof1tubulinheterodimerperkinesinheadwasused.
Thetubulinpeak(therightmostpeakvisibleinFig.
S1AandFig.
S1B)isthenentirelyshiftedtoanearlierelutionpositioninFig.
S1C.
PageS4S2.
2Dockingtubulinheterodimerstotheratkinesindimer(3KIN)crystalstructureFigureS2(below)showsthatthetubulinbindingsitesof3KINarepositionedsothatweretubulinheterodimerstobindtothisstructure,theywouldbespacedwellapartandwouldnotclashwithoneanother.
In3KIN,bothheadsareoccupiedbyADP,andbothnecklinkersaredocked.
OurdatashowthatonlyonetubulinheterodimercanbindtoakinesindimerintheabsenceofAMPPNP.
FittingofcryoEMdatasuggeststhepossibilitythatthetubulinbindingsiteinthesecondheadisphysicallymasked(below).
Furtherworkwillberequiredtodeterminethestructuralmechanismbywhichthetubulinbindingsiteonthesecondheadisblocked.
FigureS2|Dockingtubulinheterodimerstotheratkinesindimer(3KIN)crystalstructurePageS5S2.
3Dockingakinesincrystalstructureintotheparkedheadofacryo-EMstructureThemicrotubuleboundheadThecryo-EMsurfacecontourmapshowninFig.
S3isof15-pfMTsdecoratedwithdimericratkinesin-1intheabsenceoffreenucleotide(8),expectedtocorrespondtotheATP-waitingdwellstateinthewalkingmechanism.
Thismaphasaresolutionof~30.
SupplementaryFigs.
S4A-Cshowdockingofamodelcomprisingatubulinprotofilamentwithonekinesinheadbound(whitepolypeptidebackbone)intotheCryoEMmap(bluenet).
ThismodelcomplexcomprisestubulinandthemotordomainofKar3inanorientationderivedinseparateexperimentsbydockingintoahighresolutioncryo-EMmap(9).
Themaincontactwithtubulinismadebyhelixα4ofthemotordomain,aswasalsofoundforKif1A(10).
Thecrystalstructuresofkinesin-1'scanbealignedtothoseofKar3withr.
m.
s.
differencesof1.
5-3,sothetubulin-Kar3complexservesasasufficientmodelhere.
ThetetheredheadWethenconsideredthepositionofthesecond(tethered)head.
Fittingwasdonebyeyeusingoneheadoftheratkinesindimer3KIN,requiringthatthetwostrandsofthecoiledcoilneckofthedimerremainclose.
Figs.
S4A-Cshowthetetheredheadingreen.
Thisapproachyielded2possibledockingsforthesecondhead,AandB.
Boththesedockingsforthesecondmotordomainhavetheα-helicalneckofthesecondmotordomaininroughlytherightregiontopairintoacoiledcoilwiththatofthefirstmotordomain.
Fig.
S4Ashowsthefrontviewofthesetwoalternativefits.
ItisclearthatdockingAprovidesthebetterfit.
Yellowarrowspointtothestretchofhelixthatwouldformhalfofthecoiled-coilstalk.
Thedifferencesbetweenthedockingsarelessdistinctinotherviews(Fig.
S4B&S4C)sincetheoverallshapeofthemotordomainisroughlytriangular,withupto6possibledockings.
CaveatIndoingthisfitting,weconsideredthepossibilitythatthebulkofthekinesin-1motordomainmaymoverelativetoα4,ashasbeenobservedforKif1A(10,11),butnotforKar3(9).
Ifsuchamovementoccurred,itwouldnoticeablyaffectthepositionofthealphahelicalcoiledcoilstalkbutnotthespaceavailablefordockingthe2ndhead.
TheworkonKar3suggeststhatthetopoftheboundmotordomainundergoesasignificantconformationalchangeintheapostatebutthereisnocrystalstructureforthisstate.
Accordingly,thepossibilitythatthemainpartoftheheadmightshiftrelativetoalpha4limitstheprecisionofthefitting.
Specifically,thereisuncertaintyabouttheprecisepositionoftheendofthenecklinkerofthethemicrotubule-boundhead.
Nonethelesstheapproximatefitobtainedisrobust,becausetheelongatedshapeoftheheadincombinationwitharequirementforcloseappositionofthetwonecksprovidesasufficientconstraint.
PageS6SConclusionsHigherresolutionstudiesoftubulinanddimerickinesinarerequiredtoestablishthepreciseandunequivocalorientationforthesecondhead.
Itisnonethelessclearthatthemostreasonablefit(dockingA),leadstopartialocclusionofthemicrotubulebindingsurfaceofthetetheredhead,suggestingthattubulinbindingbythetetheredheadisinhibitedinthedwellstatebyparkingthetetheredheadagainsttheboundheadsoastomaskitstubulinbindingsite.
AlonsoetalSupplementaryFig.
S3.
CryoEMmapofthedwellstate(8)PageS7SAlonsoetalSupplementaryFig.
S4.
FittingthecryoEMmap.
Fig.
S4A|FrontViewDockingADockingBPageS8SFig.
S4B|SideViewDockingADockingBPageS9SFig.
S4C|TopViewDockingADockingBSupplementaryReferences1.
I.
Crevel,N.
Carter,M.
Schliwa,R.
Cross,EmboJ18,5863(1999).
2.
I.
M.
Crevel,A.
Lockhart,R.
A.
Cross,JMolBiol273,160(1997).
3.
A.
Lockhart,I.
M.
Crevel,R.
A.
Cross,JMolBiol249,763(1995).
4.
T.
Mitchison,M.
Kirschner,Nature312,232(Nov15-21,1984).
5.
A.
Davis,C.
R.
Sage,L.
Wilson,K.
W.
Farrell,Biochemistry32,8823(Aug31,1993).
6.
A.
Lockhart,R.
A.
Cross,EmboJ13,751(1994).
7.
J.
W.
Walker,G.
P.
Reid,J.
A.
McCray,D.
R.
Trentham,JAmChemSoc110,7170(1988).
8.
K.
Hirose,J.
Lowe,M.
Alonso,R.
A.
Cross,L.
A.
Amos,MolBiolCell10,2063(1999).
9.
K.
Hirose,E.
Akimaru,T.
Akiba,S.
A.
Endow,L.
A.
Amos,MolCell23,913(Sep15,2006).
10.
R.
Nitta,M.
Kikkawa,Y.
Okada,N.
Hirokawa,Science305,678(Jul30,2004).
11.
M.
Kikkawa,N.
Hirokawa,EmboJ(Aug31,2006).
tubulin

昔日数据:香港云服务器(2G防御)、湖北云服务器(100G防御),首月5折,低至12元/月

昔日数据,国内商家,成立于2020年,主要销售湖北十堰和香港HKBN的云服务器,采用KVM虚拟化技术构架,不限制流量。当前夏季促销活动,全部首月5折促销,活动截止于8月11日。官方网站:https://www.xrapi.cn/5折优惠码:XR2021湖北十堰云服务器托管于湖北十堰市IDC数据中心,母鸡采用e5 2651v2,SSD MLC企业硬盘、 rdid5阵列为数据护航,100G高防,超出防...

DiyVM:香港VPS五折月付50元起,2核/2G内存/50G硬盘/2M带宽/CN2线路

diyvm怎么样?diyvm这是一家低调国人VPS主机商,成立于2009年,提供的产品包括VPS主机和独立服务器租用等,数据中心包括香港沙田、美国洛杉矶、日本大阪等,VPS主机基于XEN架构,均为国内直连线路,主机支持异地备份与自定义镜像,可提供内网IP。最近,DiyVM商家对香港机房VPS提供5折优惠码,最低2GB内存起优惠后仅需50元/月。点击进入:diyvm官方网站地址DiyVM香港机房CN...

2021年7月最新洛杉矶CN2/香港CN2 vps套餐及搬瓦工优惠码 循环终身优惠6.58%

搬瓦工怎么样?2021年7月最新vps套餐推荐及搬瓦工优惠码整理,搬瓦工优惠码可以在购买的时候获取一些优惠,一般来说力度都在 6% 左右。本文整理一下 2021 年 7 月最新的搬瓦工优惠码,目前折扣力度最大是 6.58%,并且是循环折扣,续费有效,可以一直享受优惠价格续费的。搬瓦工优惠码基本上可能每年才会更新一次,大家可以收藏本文,会保持搬瓦工最新优惠码更新的。点击进入:搬瓦工最新官方网站搬瓦工...

akiba-online为你推荐
站酷zcool有那位知道从哪个网站能下到广告素材微信回应封杀钉钉微信大封杀"违规"了吗甲骨文不满赔偿未签合同被辞退的赔偿甲骨文不满赔偿如果合同期不满被单位辞退,用人单位是否需要赔偿蒋存祺蒋存祺的主要事迹baqizi.cc徐悲鸿到其中一张很美的女人体画dadi.tv海信电视机上出现英文tvservice是什么意思?dadi.tv1223tv影院首页地址是什么?1223tv影院在哪里可以找到?5566.com5566网址大全关键词分析关键词分析的考虑思路是怎样的,哪个数据是最重要的
北京虚拟主机 云南服务器租用 域名查询工具 浙江vps 域名服务器上存放着internet主机的 个人域名备案 lamp 荷兰服务器 hostgator 2014年感恩节 sub-process 密码泄露 php免费空间 域名转向 秒杀预告 刀片服务器的优势 爱奇艺vip免费试用7天 网通服务器 智能dns解析 云服务是什么意思 更多