DYNAMIC EVENTS IN MYOSIN DURING CONTRACTION OF MUSCLE

本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，重点关注肌动球蛋白功能的关键方面及其在过桥周期中的调节。以下具体目标将在三个项目中实现：1 .强结合的动作-S1状态：(i)验证环2在肌凝蛋白头部（S1）和肌动蛋白n端从弱结合到强结合的动作-S1状态转变中的特定作用的假设；（ii）测试序列50-58在actin - s1相互作用中对actin的作用；（iii）通过肌动蛋白和S1上的半胱氨酸突变，以及随后的交联（二硫化物和其他）和actito -S1界面的映射，测试和完善actito -S1结构模型。2。S1的动态转换和耦合：(i)绘制S1的核苷酸和肌动蛋白结合状态中S1中继区域的构象状态；（ii）通过SH1和SH2基团的修饰和交联，比较扇贝和骨骼S1中SH1-SH2螺旋的灵活性；（iii）测试和绘制S1中的几个耦合通路：在SH1-SH2螺旋、继电器区域、转换器、环路1和2以及S1上的其他位点之间。3。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）测试疏水塞（残基262-274）在肌动蛋白聚合中的作用；（iii）结晶交联的(Q41-C374) α -骨架肌动蛋白二聚体，结合到凝胶的第1段；（iv）记录动态变化对F-actin在其功能和调控中的作用。拟议的研究将结合强大的蛋白质突变替代方法与肌动球蛋白功能的溶液测量（actito -S1结合，atp酶活性，调节，体外运动性和力产生）以及通过标记，交联，荧光测量和计算建模研究F-actin， S1和actito -S1结构和动力学。提出的研究的关键组成部分是突变蛋白的制备，以及对肌凝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鉴定的心脏和骨骼肌肌病。

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.