DYNAMIC EVENTS IN MYOSIN DURING CONTRACTION OF MUSCLE

这项研究的长期目标是阐明肌肉收缩的机制。这笔赠款将支持三个项目的工作，这些项目侧重于肌动球蛋白功能的关键方面及其在跨桥周期中的调节。这三个项目将追求以下具体目标：1.强结合的Acto-S1状态：(1)测试环状2在肌球蛋白头部(S1)和肌动蛋白的N-末端从弱结合到强结合状态转变中的特定作用的假设；(2)测试Acto-S1上的序列50-58在acto-S1相互作用中的作用；(3)通过突变Acto-S1上的半胱氨酸来测试和完善acto-S1结构的模型，以及随后的交联(二硫键和其他)以及acto-S1界面的定位。Ii.S1中的动态跃迁和偶联：(I)绘制S1中继区在S1的核苷酸和肌动蛋白结合状态下的构象状态；(Ii)通过对扇贝和骨骼S1中SH1和SH2基团的修饰和交联，比较SH1-SH2螺旋在扇贝和骨骼S1中的灵活性；(Iii)测试和定位S1中的几条偶联路径：在SH1-SH2螺旋、中继区、转换器、环1和环2以及S1上的其他位置之间。F-肌动蛋白的结构、动力学和调控：(I)通过半胱氨酸酵母肌动蛋白突变体的二硫键交联和随后的模型计算，测试和完善F-肌动蛋白结构和F-肌动蛋白分子间界面的模型；(Ii)测试疏水插头(残基262-274)在肌动蛋白聚合中的作用；(Iii)结晶与明胶蛋白片段1结合的交联型(Q41-C374)α-骨骼肌动蛋白二聚体；(Iv)记录动态变化对F-肌动蛋白的功能和调控的作用。拟议的研究将结合蛋白质中强大的突变替代方法与肌动球蛋白功能的溶液测量(acto-S1结合、ATPase活性、调节、体外运动性和作用力产生)，以及通过标记、交联、荧光测量和计算模拟来研究F-肌动蛋白、S1和acto-S1的结构和动力学。这项拟议研究的关键组成部分是突变蛋白的制备，以及肌球蛋白和肌动蛋白工作的协作整合。这项工作的结果将有助于理解肌肉功能和功能障碍，特别是在越来越多的基因识别的心肌和骨骼肌病。

The long-term goal of this research is to elucidate the mechanism of muscle contraction. This grant will support work on three projects that focus on critical aspects of actomyosin function and its regulation in the cross-bridge cycle. The following specific aims will be pursued in the three projects: I. Strongly Bound Acto-S1 States: (i) to test the hypothesis for a specific role of loop 2 on myosin heads (S1) and actin's N-terminus in the transition from the weakly to strongly bound acto-S1 states; (ii) to test the role of sequence 50-58 on actin in acto-S1 interactions; (iii) to test and refine the models of the acto-S1 structure by mutations to cysteine on actin and S1, and the subsequent cross-linking (disulfide and other) and mapping of the acto-S1 interface. II. Dynamic Transitions and Couplings in S1: (i) to map the conformational states of the S1 relay region in the nucleotide and actin bound states of S1; (ii) to compare the flexibility of the SH1-SH2 helix in scallop and skeletal S1 by modifications and cross-linking of their SH1 and SH2 groups; (iii) to test and map several coupling pathways in S1: between the SH1-SH2 helix, the relay region, the converter, loops 1 and 2, and other sites on S1. III. Structure, Dynamics and Regulation of F-actin: (i) to test and refine the models of F- actin structure and the intermolecular interfaces in F-actin through disulfide cross-linkings of cysteine yeast actin mutants and the subsequent model calculations; (ii) to test the role of the hydrophobic plug (residues 262-274) in actin polymerization; (iii) to crystallize cross-linked (Q41-C374) alpha-skeletal actin dimers bound to segment 1 of gelsolin; (iv) to document the role of dynamic changes on F-actin in its function and regulation. The proposed research will combine the powerful approach of mutational substitutions in proteins with solution measurements of actomyosin function (acto-S1 binding, ATPase activity, regulation, in vitro motility, and force generation) and with the studies of F-actin, S1, and acto-S1 structure and dynamics through labeling, cross-linking, fluorescence measurements, and computational modeling. Key components of the proposed research are the preparation of mutant proteins, and the collaborative integration of the work on myosin and actin. The results of this work will contribute to the understanding of muscle function and malfunction, especially in the growing number of genetically identified cardiac and skeletal muscle myopathies.