Molecular Dynamics of Muscle Contraction

描述（由申请人提供）：该项目的目标是确定肌动球蛋白结构动力学在肌肉产生力的分子机制中的作用。重点放在使用高分辨率和时间分辨（TR）光谱来测试和修订肌球蛋白和肌动蛋白功能相互作用的详细机制模型。(a)肌凝蛋白和肌动蛋白的晶体结构和电子显微图为肌动蛋白的结构动力学提供了高分辨率的模型，这必须通过功能性蛋白质复合物的位点定向光谱进行测试和修正，使用计算模拟将模型与实验联系起来。(b)弱到强（W-S）的转变是基本的，在肌动蛋白和肌凝蛋白中都发生。(c)为了了解这些转变的物理基础，肌动蛋白和肌凝蛋白的弱和强状态必须以动态和无序以及结构为特征。有五个目标：(1)发展改进的肌球蛋白和肌动蛋白结构动力学的位点定向光谱分析方法。最近获得或开发的新光谱仪将该项目提升到一个新的技术水平：脉冲电子顺磁共振（EPR），高频EPR和高通量脉冲荧光，在稳态和瞬态生化条件下进行，为测量角度和距离以及解决它们之间的结构状态和转换提供了新的机会。光谱学将与计算模拟、x射线晶体学和电子显微镜（EM）的合作研究相协调。(2)研究肌球蛋白催化结构域的结构动力学，测试和修正机制模型。以三个不同的区域（力产生区、肌动蛋白结合区、核苷酸袋区）为中心，在瞬态条件下进行实验，分析运动部件在空间和时间上的协调。功能突变将用于区分弱和强状态。(3)研究轻链（LC）结构域的结构动力学，以表征肌球蛋白调节轻链（RLC） n端磷酸化结构域（PD）先前未知的结构，并确定其受磷酸化、ATP和肌动蛋白影响的内部和全局动力学。(4)与肌凝蛋白（包括肌凝蛋白异构体和功能突变体）的强、弱相互作用会干扰肌动蛋白的整体和内部结构动力学，从而将结构动力学与功能联系起来。(5)利用探针在两种蛋白上绘制肌动球蛋白界面，利用电子显微镜将数据整合到弱态和强态的结构模型中。这项工作对于理解肌肉功能至关重要，这里产生的技术和概念已经被其他地方使用，由这个小组和其他人，在分子水平上提供对肌肉功能障碍和治疗的见解。更一般地说，在这个项目中吸取的经验教训适用于细胞运动的生物物理学中的广泛问题。公共卫生相关性：拟议的研究汇集了从分子生物学到生物化学再到生物物理学的强大技术组合，以解决肌肉中产生力的分子机制。这项工作对理解肌肉功能至关重要，这里产生的技术正被应用于其他地方，以提供肌肉功能障碍的分子洞察力。更一般地说，这个定义良好的系统可以作为研究分子动力学和运动蛋白相互作用的模型，我们正在开发的方法应该在分析该领域的广泛问题中被证明是有效的。

DESCRIPTION (provided by applicant): The goal of this project is to determine the role of actomyosin structural dynamics in the molecular mechanism of force generation in muscle. Emphasis is placed on the use of high-resolution and time- resolved (TR) spectroscopy to test and revise detailed mechanistic models for the functional interaction of myosin and actin. Several hypotheses inform all aims: (a) Crystal structures and electron micrographs of myosin and actin provide high-resolution models for the structural dynamics of actomyosin, which must be tested and revised by site-directed spectroscopy in functional protein complexes, using computational simulations to connect models with experiment. (b) The weak-to-strong (W-S) transition is fundamental and occurs in both actin and myosin. (c) To understand the physical basis of these transitions, the weak and strong states of actin and myosin must be characterized by dynamics and disorder as well as structure. There are five aims: (1) Development of improved methods for site-directed spectroscopic analysis of myosin and actin structural dynamics. New spectrometers, recently acquired or developed, have raised this project to a new technological level: Pulsed electron paramagnetic resonance (EPR), high-frequency EPR, and high- throughput pulsed fluorescence, performed under both steady-state and transient biochemical conditions, opens new opportunities to measure angles and distances, and to resolve structural states and transitions between them. Spectroscopy will be coordinated with collaborative studies of computational simulations, x- ray crystallography and electron microscopy (EM). (2) Structural dynamics of the myosin catalytic domain will be probed to test and revise mechanistic models. Focusing on three distinct regions (force-generating, actin-binding cleft, nucleotide pocket) and performing experiments under transient conditions, coordination of motor parts will be analyzed in space and time. Functional mutations will be used to distinguish weak and strong states. (3) Structural dynamics of the light chain (LC) domain will be probed to characterize the previously unknown structure of the N-terminal phosphorylation domain (PD) of the myosin regulatory light chain (RLC), and to determine its internal and global dynamics as affected by phosphorylation, ATP, and actin. (4) Actin structural dynamics, both global and internal, will be perturbed by weak and strong interaction with myosin, including myosin isoforms and functional mutants, to correlate structural dynamics with function. (5) The actomyosin interface will be mapped by probes on both proteins, and EM will be used to integrate the data into structural models for the weak and strong states. This work is of fundamental importance for understanding muscle function, and the technology and concepts generated here are already being used elsewhere, by this group and others, to provide insight into muscle malfunction and therapy at the molecular level. More generally, the lessons learned in this project are applicable to a wide range of problems in the biophysics of cellular movement. PUBLIC HEALTH RELEVANCE: The proposed research brings together a powerful combination of techniques, from molecular biology to biochemistry to biophysics, to solve the molecular mechanism of force generation in muscle. This work is of fundamental importance for understanding muscle function, and the technology generated here is being applied elsewhere to provide molecular insight into muscle malfunction. More generally, this well-defined system serves as a model for studying the role of molecular dynamics and interactions in motor proteins, and the approaches we are developing should prove effective in the analysis of a wide range of problems in this field.