MOLECULAR ANGLE TRANSITIONS IN MUSCLE

产生肌肉缩短的力量发生在 肌纤维中的三磷酸腺苷的水解酶。将数据转换为 从三磷酸腺苷水解物中释放出来的化学能变成机械功 (能量转导)涉及肌球蛋白的结构变化 过桥。这些变化的本质以及它们如何使肌肉工作 是肌肉研究的中心问题。建议的目的是 实验旨在阐明肌动球蛋白相互作用的机制。 以及它如何通过确定相对的肌肉缩短 收缩过程中肌球蛋白和肌动蛋白的运动。我们调查僵化 肌球蛋白的体内或分子内运动的方向报告 化学修饰分子上选定侧链的非本征探针 交叉桥，和，荧光偏振(FP)和/或电子 顺磁共振(EPR)波谱检测探头方向。 探针(光学或EPR)对蛋白质旋转的敏感性取决于 蛋白质固定参照物中探针定位的批判性研究 由于方向检测技术中的模糊性而导致的帧。我们可能会 通过组合来自不同探测器的数据来减少模糊性 使用已证实的形式相对于蛋白质进行不同的定向 绘制组装中蛋白质的全球旋转图谱。我们将延长这一期限 方法：(I)通过关联下列数据来调查局部顺序变化 空间分离位置的探测器，(Ii)提高角分辨率 探头角度分布是否达到理论极限，以及(Iii) 介绍和研究了一类新的探头的结构 发光和顺磁(L/P)。为阐明其发病机制 导致肌肉缩短的蛋白质相互作用我们将依赖于我们的 稳态跨桥顺序的分辨率越来越高的地图等 我们区分局部秩序变化和全球变化的能力。fp 和EPR有时间分辨的类似物来研究旋转动力学 在亚毫秒级的时间域上跨越桥梁。极化 荧光光漂白恢复(PFPR)诱导定向的子集 全套荧光探针的光漂白探针 强烈的偏振光脉冲。漂白区域用来监测 衰减的偏振光和荧光恢复被观察为 未漂白的荧光团旋转，它们的偶极子与 监控光的偏振。时间分辨EPR(TEPR)使用L/P 在光照射下形成自由基的探针。一场激烈的 偏振光脉冲导致定向子集的光选择 激进分子。TEPR信号随着定向自旋的旋转而改变。为 PFPR和TEPR信号强度松弛到其稳态值 探头移动性的时间特性。PFPR和TEPR调查探针 在三个转动自由度中运动。这两种技术的结合 静态和动态探测顺序信息提供了全面的 肌动球蛋白相互作用的描述。

The generation of force to produce muscle shortening takes place during the hydrolysis of ATP in a muscle fiber. The mechanism of converting the chemical energy liberated from ATP hydrolysis into mechanical work (energy transduction) involves structural changes in the myosin cross-bridge. The nature of these changes and how they make muscle work is the central question in muscle research. The purpose of the proposed experiments is to elucidate the mechanism of the actomyosin interaction and how it pertains to muscle shortening by determining the relative movements of myosin and actin during contraction. We investigate rigid body or intramolecular movements of myosin by using direction reporting extrinsic probes that chemically modify selected side chains on the cross-bridge, and, fluorescence polarization (FP) and/or electron paramagnetic resonance (EPR) spectroscopy to detect probe orientation. The sensitivity of probes (optical or EPR) to protein rotation depends critically on the orientation of the probe in the protein-fixed reference frame due to ambiguity in the orientation detection techniques. We may reduce the ambiguity by combining data from different probes that are variously oriented relative to the protein using a proven formalism for mapping global rotations of proteins in assemblies. We will extend this method by: (i) investigating local order changes by correlating data from probes of spatially separated sites, (ii) boosting the angular resolution of the probe angular distributions to the theoretical limit, and (iii) introducing and studying the structure of a new class of probes that are luminescent and paramagnetic (L/P). To elucidate the mechanism of protein interaction that results in muscle shortening we will rely on our increasingly well resolved maps of steady-state cross-bridge order and on our ability to distinguish local order changes from global changes. FP and EPR have time-resolved analogues to investigate rotational dynamics of cross-bridges on the submillisecond time domain. Polarized fluorescence photobleaching recovery (PFPR) induces an oriented subset of photobleached probes into the total set of fluorescent probes by an intense pulse of polarized light. The bleached region is monitored with attenuated polarized light and the fluorescence recovery is observed as unbleached fluorophores rotate and their dipoles align with the polarization of the monitoring light. Time-Resolved EPR (TEPR) uses L/P probes that form free radicals when illuminated with light. An intense pulse of polarized light causes the photoselection of an oriented subset of radicals. The TEPR signal changes as the oriented spins rotate. For PFPR and TEPR the signal intensity relaxes to its steady-state value in a time characteristic of probe mobility. PFPR and TEPR investigate probe movement in the three degrees of rotational freedom. The combination of static and dynamic probe order information provides a comprehensive description of the actomyosin interaction.