MYOFIBRILLAR STRUCTURE OF STRIATED MUSCLE

横纹肌的肌原纤维结构

• 批准号: 3154806
• 负责人: MICHAEL KAY REEDY
• 金额: $ 20.89万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-05-01 至 1996-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3154806
• 关键词: Insecta; X ray crystallography; actins; adenosine triphosphate; calcium; chimeric proteins; computer simulation; conformation; electron microscopy; flash photolysis; fluorescence microscopy; freeze substitution; gelsolin; image processing; laboratory rabbit; muscle contraction; muscle relaxation; muscle tension; mutant; myofibrils; myosins; nucleotide analog; protein structure; sarcomeres; site directed mutagenesis; striated muscles; structural biology

Our main goal is to identify the structural changes that accompany the myosin power stroke and cause force generation in muscle, to a resolution of about 5 nm in space and about 5 ms in time. To correlate the structure, mechanics and timing of crossbridge action in the highly ordered insect flight muscle (IFM) of Lethocerus, we will coordinate electron microscopy, X-ray diffraction and physiological measurements. Rapid structural transitions will be time-resolved by quick freezing, and examined by thin-section EM and image reconstruction of freeze- substituted muscle, supported by time-slicing synchrotron X-ray diffraction. Concerted crossbridge transitions will be triggered by photolysis of "caged" (inert photolabile precursors of) substrate or signal molecules, and also by sudden length steps that produce stretch-activation in IFM. The steady-state equilibrium structures of quick-frozen IFM fibers in rigor, relaxed and isometrically contracting states will be established first, to be followed by studies of rapid transitions. We will time-resolve structural changes of the crossbridge cycle by EM of fibers quick-frozen at 15, 30, 50, 100 and 300 ms after photochemical triggering of the rigor-to-relaxed and rigor-to-active transitions. Stretch-activation of the relaxed-to-active transition will be similarly time-resolved. Rigor contractions will be a special focus, if we can achieve rapid single-turnover relaxed-to-rigor transitions. X-ray diffraction including time-slicing studies at synchrotron X-ray sources will characterize the same photolytically or stretch-activated transitions. We will try to capture quicker and better synchronized transitions by quick-freezing contracting fibers during force transients that follow sudden length steps. Quick release may synchronize a quick-recovery power stroke. Quick stretch should drag attached bridges "backwards" into their highest force-producing state. Structure of "pre-force" attached crossbridge state(s) will be explored by EM and X-ray comparison of fibers that develop stiffness but little or no force/shortening in, high Ca2+ after equilibration with glycol-AMPPNP, ATPgammaS, ADPAIF3, PrNANTP and possibly ADP-Vi. We will collaborate in seeking spin-label strategies and probes that may report closer agreement with EMs and X-ray about orientations of the bulk of averaged myosin head mass, apparently uniform (at 90 degrees) in relaxed, and multiple (at 48 degrees, 77 degrees and 90 degrees) in rigor. Collaborative mutational analysis of actin-crossbridge interaction in site-directed actin-mutant Drosophila flight muscle will include our ultrastructural analysis and participation in in vitro motility studies, and 3D reconstructions of normal and reverse rigor chevrons when possible.

我们的主要目标是确定伴随 肌球蛋白动力冲程和导致肌肉力量的产生，以解决 在空间上约为5 nm，在时间上约为5 ms。 来关联 结构，力学和时间的交叉桥行动，在高度 订购昆虫飞行肌肉（IFM）的Lethocerus，我们将协调 电子显微镜、X射线衍射和生理测量。 快速的结构转变将通过快速冷冻来进行时间分辨， 用薄层电镜和冷冻图像重建技术进行检查， 替代肌肉，由时间切片同步加速器X射线支持 衍射法 协调的跨桥过渡将由 “笼状”底物（的惰性光不稳定前体）的光解，或 信号分子，也可以通过突然的长度步骤， IFM中的牵张激活。 的稳态平衡结构 速冻IFM纤维僵硬，松弛和等距收缩 国家将首先建立，其次是快速的研究， 过渡。 我们将对天桥的结构变化进行时间解析 在15、30、50、100和300 ms后通过EM循环快速冷冻的纤维 光化学触发的严格到放松和严格到活动 过渡。 从弛豫到激活的转变的拉伸激活将 时间分辨相似。 僵硬性宫缩将是一个特别的重点， 如果我们能实现快速的单次翻转从放松到僵硬的转变。 X射线衍射，包括同步加速器X射线下的时间切片研究 来源将表征相同的光解或拉伸激活 过渡。 我们将努力捕捉更快更好的同步 在力瞬变期间通过快速冻结收缩纤维的转变 在突然的长步之后。 快速释放可以同步 快速恢复动力冲程。 快速拉伸应拖动附加的桥梁 “倒退”到最高的生产力状态。 结构 EM将探索“预力”连接的横桥状态， X射线比较纤维的发展刚度，但很少或没有 力/缩短，用乙二醇-AMPPNP平衡后的高Ca 2+， ATP γ S、ADPAIF 3、PrNANTP和可能的ADP-Vi。 我们将合作， 寻求自旋标签战略和探针，可能报告更密切的协议 用EM和X-射线关于平均肌球蛋白头部的大部分方向 质量，明显均匀（在90度）在放松，和多个（在48 度、77度和90度）的僵硬。 协同突变 肌动蛋白定点突变体中肌动蛋白-交联桥相互作用的分析 果蝇飞行肌肉将包括我们的超微结构分析， 参与体外运动研究，以及3D重建， 正常和反向僵硬的人字形。