Crystallization and structural study of muscle actomyosin complex

研究表明，用间马来酰亚胺苯甲酸n -羟基琥珀酰亚胺酯（MBS）处理g -肌动蛋白可以阻断盐存在下g -肌动蛋白的聚合。MBS-actin保留了与肌凝蛋白头结合（并产生化学交联）的能力[Bettache， N., Bertrand， R., & Kassab， R.(1989) proc . natl . academy . u.s.a。86, 6028-6032；J.Biochem Arata t(1991)。109年,335 - 340]。本研究通过蛋白水解敏感性和化学交联进一步研究了MBS-actin与肌球蛋白亚片段1（S1）之间的相互作用。2摩尔mbs -肌动蛋白单体与1摩尔肌凝蛋白头或S1结合。MBS-actin与S1的首次结合对S1重链的27kDa/50kDa连接有较强的保护作用，对S1重链的50kDa/20kDa连接也有较弱的保护作用。第二种结合对50kDa/20kDa结的保护作用更强。通过蛋白水解分析，确定了第一和第二结合位点的解离常数分别为1-2和4-5 × 10^<-6>M。ATP将这些结合削弱了10倍以上。MBS-actin在第一个结合位点与S1交联，得到的复合物在电泳凝胶上以180kDa的速度迁移。在ATP或ADP存在的情况下，一个140 kDa的复合物与180 kDa的复合物一起产生。目前正在进行电子显微镜研究，以检查单体mbs - g -肌动蛋白- hmm复合物，这些复合物是在没有和存在ATP的情况下通过交联产生的。初步实验表明，在没有ATP的情况下产生的acto-HMM分子的肌动蛋白结合位点位于距离头杆连接处14-15 nm处。相比之下，在ATP存在下产生的acto-HMM分子显示，其位置距离结约13 nm，略靠近颈部区域。

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.