MYOFIBRILLAR STRUCTURE OF STRIATED MUSCLE

我们的主要目标是确定随之而来的结构性变化 肌球蛋白为中风提供动力，并在肌肉中产生力量，以解决 在空间上约5纳米，在时间上约5毫秒。若要将 高寒地区跨桥作用的结构、力学和时机 有秩序的昆虫飞翔肌肉(IFM)，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冻结来及时解决，以及 冰冻切片电镜片检查及图像重建 替代肌肉，由时间切片同步加速器X射线支持 绕射。协调一致的跨桥过渡将由 “笼状”(惰性耐光前驱体)底物或 信号分子，以及产生的突如其来的长度台阶 IFM中的拉伸激活。系统的稳态平衡结构 僵硬、松弛和等长收缩的速冻IFM纤维 首先将建立国家，随后将研究快速 过渡。我们将及时解决立交桥的结构变化 15、30、50、100和300ms速冻纤维的EM循环 从严格到放松和从严格到活跃的光化学触发 过渡。从松弛到活跃的转变的拉伸激活将 同样要有时间分辨能力。僵硬的宫缩将是一个特别的焦点， 如果我们能够实现快速的单周转，从宽松到严谨的过渡。 包括同步加速器X射线时间切片研究的X射线衍射 来源将表征相同的光解或拉伸激活 过渡。我们将努力以更快的速度和更好的同步 力瞬变过程中快速冻结收缩纤维的转变 紧跟在突如其来的长步之后。快速释放可能会同步 快速恢复动力冲程。快速伸展应拖拽连接的桥梁 “倒退”进入他们最高的武力生产状态。的结构 《预紧力》附中跨桥状态(S)将由EM和 出现僵硬但很少或没有僵硬的纤维的X射线比较 用力/缩短，与乙二醇AMPPNP平衡后高钙， ATPGammaS、ADPAIF3、PrNANTP，可能还有ADP-Vi。我们将在以下方面进行合作 寻求可能报告更接近一致的自旋标签策略和调查 平均肌球蛋白头大块定位的电子显微镜和X射线检查 质量，明显均匀(在90度)，松弛，多重(在48度 度、77度和90度)严谨。协作性突变 肌动蛋白定点突变体的肌动蛋白-交叉桥相互作用分析 果蝇的飞行肌肉将包括我们的超微结构分析和 参与体外运动研究和三维重建 在可能的情况下，正常的和反向的僵硬的人字形。

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.