MOLECULAR ANGLE TRANSITIONS IN MUSCLE

肌肉中的分子角转变

• 批准号: 2079495
• 负责人: Thomas P Burghardt
• 金额: $ 21.05万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-01-01 至 1996-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2079495
• 关键词: actins; benzopyrans; bioenergetics; biophysics; computer simulation; conformation; crosslink; dyes; electron spin resonance spectroscopy; fluorescence polarization; fluorescent dye /probe; hydrolysis; intermolecular interaction; laboratory rabbit; luminescence; mechanical stress; microfilaments; molecular dynamics; muscle contraction; muscle stress; myosins; protein reconstitution; protein structure; stable isotope; thiols; time resolved data

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.

产生肌肉缩短的力发生在 肌纤维中 ATP 的水解。 转换机制 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 过桥。 这些变化的本质以及它们如何使肌肉发挥作用 是肌肉研究的中心问题。 拟议的目的 实验的目的是阐明肌动球蛋白相互作用的机制 以及通过确定相对值来了解它与肌肉缩短的关系 收缩期间肌球蛋白和肌动蛋白的运动。 我们调查刚性 通过使用方向报告来了解肌球蛋白的身体或分子内运动 化学修饰选定侧链的外在探针 跨桥、荧光偏振 (FP) 和/或电子 顺磁共振 (EPR) 光谱检测探针方向。 探针（光学或 EPR）对蛋白质旋转的敏感性取决于 关键在于蛋白质固定参考中探针的方向 由于方向检测技术的模糊性而导致的框架。 我们可能 通过组合来自不同探针的数据来减少歧义 使用经过验证的形式相对于蛋白质进行不同的定向 绘制组件中蛋白质的全局旋转。 我们将延长此 方法如下：（i）通过关联来自的数据来调查当地秩序的变化 空间分离位点的探针，(ii) 提高角分辨率 探头角度分布达到理论极限，以及 (iii) 介绍并研究一类新型探针的结构 发光和顺磁（L/P）。 为了阐明其机制 导致肌肉缩短的蛋白质相互作用，我们将依靠我们的 稳态跨桥顺序图的解析度越来越好 我们区分局部秩序变化和全局变化的能力。 FP 和 EPR 具有时间分辨类似物来研究旋转动力学 亚毫秒时域上的跨桥。 偏振 荧光光漂白恢复 (PFP​​R) 诱导定向子集 光漂白探针进入荧光探针的总组 强烈的偏振光脉冲。 漂白区域受到监测 衰减的偏振光和荧光恢复被观察为 未漂白的荧光团旋转并且它们的偶极子与 监测光的偏振。 时间分辨 EPR (TEPR) 使用 L/P 在光照射下形成自由基的探针。 强烈的 偏振光脉冲导致定向子集的光选择 的部首。 TEPR 信号随着定向自旋的旋转而变化。 为了 PFPR 和 TEPR 中信号强度松弛至其稳态值 探针迁移率的时间特性。 PFPR 和 TEPR 调查探针 三个旋转自由度的运动。 的组合 静态和动态探针顺序信息提供了全面的 肌动球蛋白相互作用的描述。