MOLECULAR ANGLE TRANSITIONS IN MUSCLE

产生肌肉缩短的力发生在 肌纤维中ATP的水解。 转换的机制 ATP水解释放的化学能转化为机械功 （能量转导）涉及肌球蛋白的结构变化， 跨桥 这些变化的性质以及它们如何使肌肉工作 是肌肉研究的核心问题 建议的目的 实验旨在阐明肌动球蛋白相互作用的机制 以及它是如何通过确定肌肉缩短的相对 收缩时肌球蛋白和肌动蛋白的运动。 我们调查刚性 通过使用方向报告的肌球蛋白的体或分子内运动 化学修饰所选侧链的外来探针 交叉桥和荧光偏振（FP）和/或电子 顺磁共振（EPR）光谱以检测探针取向。 探针（光学或EPR）对蛋白质旋转的灵敏度取决于 关键在于探针在蛋白质固定参照物中的方向 由于方向检测技术中的模糊性， 我们可能 通过组合来自不同探测器的数据来减少模糊性， 相对于蛋白质的不同取向，使用经证实的形式主义， 绘制蛋白质组装体的全局旋转。 我们会延长这个 方法通过：（i）通过将来自 空间上分离的站点的探针，（ii）提高角分辨率 探头角分布的理论极限，以及（iii） 介绍和研究一类新的探针的结构， 发光和顺磁（L/P）。 为了阐明 蛋白质的相互作用，导致肌肉缩短，我们将依靠我们的 越来越好地解决地图的稳态跨桥秩序和 我们区分局部秩序变化和全局变化的能力。 FP 和EPR有时间分辨的类似物来研究旋转动力学 在亚毫秒时间域上的交叉桥。 极化 荧光光漂白恢复（PFPR）诱导了一个定向的子集， 将光漂白的探针插入全部荧光探针组中， 强烈的偏振光脉冲。 漂白区域用 衰减的偏振光，并且观察到荧光恢复为 未漂白的荧光团旋转，并且它们的偶极子与荧光团对准。 监测光的偏振。 时间分辨EPR（TEPR）使用L/P 在光照下形成自由基的探针。 强烈的 偏振光脉冲引起定向子集的光选择 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， 探针迁移率的时间特性。 PFPR和TEPR调查探针 在三个旋转自由度上的运动。 的组合 静态和动态探头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.