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Crystallization and structural study of muscle actomyosin complex

肌肉肌动球蛋白复合物的结晶和结构研究

基本信息

批准号：
04680269
负责人：
ARATA Toshiaki
金额：
$ 1.28万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

It was shown that polymerization of G-actin in the presence of salts is blocked by treatment of G-actin with m-maleimidebenzoic acid N-hydroxysuccinimide ester(MBS)]. MBS-actin retains the ability to bind(and make chemical crosslinks)to myosin head [Bettache, N., Bertrand, R., & Kassab, R.(1989)Proc.Natl.Acad.Sci.U.S.A.86, 6028-6032 ; Arata, T.(1991)J.Biochem.109, 335-340]. Here, the interaction between MBS-actin and myosin subfragment 1(S1)has been further studied by proteolytic susceptibility and chemical crosslinking. Two moles of MBS-actin monomers bound to 1 mole of myosin heads or S1. The first binding of MBS-actin to S1 strongly protected a 27kDa/50kDa junction of S1 heavy chain from trypsin digestion and also weakly protected a 50kDa/20kDa junction. The second binding protected a 50kDa/20kDa junction more strongly. From this proteolytic analysis, the dissociation constants for the first and second binding sites were determined to be 1-2 and 4-5 x 10^<-6>M, respectively. ATP weakened these bindings more than 10-fold. MBS-actin was crosslinked by MBS to S1 at the first binding site and the resulting complex was migrating at 180kDa on electrophoretic gels. In the presence of ATP or ADP, an 140 kDa complex was produced together with the 180 kDa complex.The electron-microscopic study is now in progress to examine the monomeric MBS-G-actin-HMM complexes which have been produced by crosslinking in the absence and presence of ATP.Preliminary experiment showed that the location of actin binding site of the acto-HMM molecules produced in the absence of ATP is 14-15 nm away from the head-rod junction. In contrast, the acto-HMM molecules produced in teh presence of ATP showed that the location is about 13 nm from the junction, slightly more proximal to the neck region.

结果表明，在盐的存在下，G-肌动蛋白的聚合被M-马来酰亚胺苯甲酸N-羟基琥珀酰亚胺酯（MBS）处理的G-肌动蛋白阻断。MBS-肌动蛋白保留了与肌球蛋白头结合（并进行化学交联）的能力[Bettache，N.，贝特朗河，和R.卡萨布。（1989）Proc.Natl.Acad.Sci.U.S.A.86，6028-6032 ;阿拉塔，T.（1991）J.Biochem.109，335-340]。在这里，MBS-肌动蛋白和肌球蛋白亚片段1（S1）之间的相互作用已被进一步研究的蛋白水解敏感性和化学交联。2摩尔MBS-肌动蛋白单体与1摩尔肌球蛋白头或S1结合。MBS-肌动蛋白与S1的第一次结合强烈地保护S1重链的27 kDa/50 kDa连接不受胰蛋白酶消化，并且也微弱地保护50 kDa/20 kDa连接。第二种结合更强地保护50 kDa/20 kDa连接。根据该蛋白水解分析，确定第一和第二结合位点的解离常数分别为1-2和4-5 × 10 - 4 <-6>M。ATP使这些结合减弱了10倍以上。MBS-actin在第一个结合位点与S1交联，所得复合物在电泳凝胶上以180 kDa迁移。在ATP或ADP的存在下，在无ATP和有ATP条件下，MBS-G-actin-HMM交联形成的单体复合物，其肌动蛋白结合位点的位置分别为14- 18 - 20-距离头杆连接处15 nm。相比之下，在ATP存在下产生的acto-HMM分子显示位置距离连接处约13 nm，稍微更接近颈部区域。