The Mechanism of Myosin ATPase Actin Activation

描述（由申请人提供）：肌动蛋白和肌球蛋白的相互作用是肌肉收缩和肌肉力量产生的基础，也是细胞运动和细胞内交通的基础。在肌凝蛋白II中，当肌凝蛋白改变其构象并推动肌动蛋白丝相对于肌肉中的粗丝或沿着细胞中的肌动蛋白细胞骨架转移货物时，肌动蛋白结合启动动力冲程，这是一个单一的力产生事件。肌动蛋白-肌球蛋白相互作用调节肌动蛋白ATP酶活性，提高ATP水解产物释放的速率。本项目的目的是确定肌球蛋白atp酶肌动蛋白激活的机制。我们假设肌动蛋白通过调节肌凝蛋白发力区域的接力环-接力螺旋相互作用来调节动力冲程。肌动蛋白结合改变了继电环的构象，使继电环的螺旋结构不稳定，诱发了继电环的发生。我们将在肌凝蛋白- ATP和肌凝蛋白-肌动蛋白相互作用过程中测量接力螺旋的构象，使用新开发的方法，基于荧光和自旋探针的定点标记，脉冲电子顺磁共振和瞬态时间分辨荧光法，两种互补的高分辨率光谱技术。正如我们之前所展示的，这些突变和标记不影响肌球蛋白的功能。我们将在接力环中引入功能性突变来干扰肌动蛋白的激活，并详细研究激活机制，分析肌动蛋白激活过程中肌球蛋白的构象和这种构象变化的时间。有两个目标：目标1。肌凝蛋白中肌动蛋白激活力产生的技术进展。这是实现我们目标的技术基础。我们将使用我们以前开发的方法来测量继电器螺旋构象变化的动力学。我们将升级我们目前的瞬态时间分辨荧光计与3注射器/2混合器停止流动装置。首先，肌凝蛋白将在短暂的动力冲程前状态下与ATP快速混合制备。然后将制备好的肌凝蛋白与肌动蛋白快速混合，启动肌动蛋白-肌凝蛋白相互作用。肌动蛋白激活的动力行程动力学将通过瞬时时间分辨荧光法监测继电器螺旋的构象变化。我们将回答这个问题：力产生的时间是什么？它与核苷酸结合和水解产物释放有什么关系？目标2。肌凝蛋白atp酶肌动蛋白活化的机制。利用脉冲电子顺磁共振和瞬态时间分辨荧光法研究功能性肌球蛋白突变体的接力螺旋构象及其瞬态结构动力学。我们将回答两个问题：接力环是肌动蛋白激活肌凝蛋白的调节元件吗？这种激活的机制是什么？这项研究对于从亚分子水平上理解肌肉功能和功能障碍的机制具有重要意义。了解这些机制将有助于合理设计肌肉功能障碍治疗，以及更好地理解肌肉收缩的机制。

DESCRIPTION (provided by applicant): Interaction of actin and myosin is the basis of muscle contraction and force generation in muscle, as well as the basis of cellular motion and intracellular traffic. In myosin II, actin binding initiates the powerstroke, a single force-generating event, when myosin changes its conformation and propels actin filament relative to thick filament in muscle or transfers cargo along actin cytoskeleton in cells. Acto-myosin interaction modulates myosin ATPase activity, enhancing the rate of ATP hydrolysis products release. The goal of this project is to determine the mechanism of myosin ATPase actin activation. We hypothesize that the powerstroke is regulated by actin via modulation of the relay loop - relay helix interaction in the force generating region of myosin. Actin binding changes the relay loop conformation and destabilizes the relay helix in the pre- powerstroke, inducing the powerstroke. We will measure the conformation of the relay helix during myosin- ATP and myosin-actin interaction, using newly developed assay, based on site-directed labeling with fluorescent and spin probes, pulsed electron paramagnetic resonance and transient time-resolved fluorimetry, two complementary high-resolution spectroscopic techniques. As we have shown previously these mutations and labeling do not affect myosin function. We will introduce functional mutations into the relay loop to perturb actin activation, and study activation mechanism in detail, analyzing myosin conformation and timing of this conformational change during actin activation. There are two Aims: Aim 1. Technology development to study actin activation of force production in myosin. This is a technical basis to achieve our goal. We will use our previously developed approach to measure kinetics of the relay helix conformational change. We will upgrade our current transient time-resolved fluorimeter with 3 syringe/2mixer stopped flow apparatus. First, myosin will be prepared in the transient pre-powerstroke state by rapid mixing with ATP. Then we will rapidly mix prepared myosin with actin to initiate acto-myosin interaction. The kinetics of actin activated powerstroke will be monitored via conformational change of the relay helix by transient time- resolved fluorimetry. We will answer the question: what is the timing of the force generation and how is it related to nucleotide binding, and products of hydrolysis release? Aim 2. The mechanism of myosin ATPase actin activation. The relay helix conformation and its transient structural dynamics in functional myosin mutants will be studied using pulsed electron paramagnetic resonance and transient time-resolved fluorimetry. We will answer two questions: is the relay loop a regulating element in actin activation of myosin? What is the mechanism of this activation? This study is of fundamental importance for understanding the mechanism of muscle function and muscle malfunction on submolecular level. Knowledge of these mechanisms will allow rational design of muscle malfunction treatment, as well as better understanding of the mechanism of muscle contraction. PUBLIC HEALTH RELEVANCE: The ultimate goal of the project is to understand how the force is generated in muscle. In the proposed research, we will study how myosin, a molecular motor, is activated by actin for the force production. To reach the goal, we will combine molecular biology, biochemistry and biophysical spectroscopy. This study is of fundamental importance for understanding the mechanism of muscle function and muscle malfunction on submolecular level. Knowledge of this mechanism will allow rational design of muscle malfunction treatment, as well as better understanding of the mechanism of muscle contraction.