The Mechanism of Myosin ATPase Actin Activation

肌球蛋白 ATP 酶肌动蛋白激活机制

基本信息

批准号：
7939094
负责人：
YURI NESMELOV
金额：
$ 40.55万
依托单位：
UNIVERSITY OF NORTH CAROLINA CHARLOTTE
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-04-01 至 2013-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7939094
关键词：
ATP Hydrolysis ATP phosphohydrolase Actins Actomyosin Address Affect Binding Binding Sites Biochemistry Biological Assay Cells Computer Simulation Cytoskeleton Data Deer Dictyostelium discoideum Disease Electron Spin Resonance Spectroscopy Elements Event Fluorescence Resonance Energy Transfer Generations Goals Heart Home environment Hydrolysis Hypertrophic Cardiomyopathy Indium Kinetics Knowledge Label Left Link Measures Microfilaments Modeling Molecular Biology Molecular Conformation Molecular Motors Monitor Motion Motor Muscle Muscle Contraction Muscle function Mutagenesis Mutation Myosin ATPase Myosin Type II Nucleotides Optics Physiologic pulse Production Proline Relative (related person)Research Resolution Site Site-Directed Mutagenesis Spectrum Analysis Syringes Takeda brand of pioglitazone hydrochloride Techniques Thick Filament Time base conformational conversion design instrumentation mutant public health relevance research study stroke recovery technology development trafficking

项目摘要

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.

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肌肉收缩和力量产生的基础，以及细胞运动和细胞内交通的基础。在肌球蛋白II中，肌动蛋白结合引发了肌球蛋白改变其构象并推动肌动蛋白丝相对于肌肉中的厚细丝或沿细胞中肌动蛋白细胞骨架的货物转移时，肌动蛋白的结合引发了肌动蛋白。肌动蛋白相互作用调节肌球蛋白ATPase活性，从而提高了ATP水解产物释放的速率。该项目的目的是确定肌球蛋白ATPase肌动蛋白激活的机制。我们假设通过肌动蛋白调节继电器环 - 继电器螺旋相互作用在肌球蛋白的力区域中的相互作用来调节动力。肌动蛋白的结合改变了继电器循环构象，并破坏了预击中的继电器螺旋，从而诱导了势力。我们将使用新开发的测定法测量继电器螺旋在肌球蛋白ATP和肌球蛋白 - 肌动蛋白相互作用期间的构象，该测定法基于荧光和自旋探针，脉冲电子磁共振谐振和瞬时时间分辨的荧光术，两种互补的高分辨率高分辨率高分辨率光谱技术。如前所述，这些突变和标记不会影响肌球蛋白功能。我们将在接力循环中引入功能突变，以详细研究肌动蛋白激活，并详细研究激活机制，分析肌动蛋白激活期间这种构型变化的肌球蛋白构象和时机。有两个目的：目标1。研究肌球蛋白中力产生的肌动蛋白激活的技术开发。这是实现我们目标的技术基础。我们将使用先前开发的方法来测量继电器螺旋构象变化的动力学。我们将使用3个注射器/2Mixer停止流动设备来升级当前的瞬时时间分辨荧光表。首先，肌球蛋白将通过与ATP快速混合在瞬态前击球状态下制备。然后，我们将迅速将制备的肌球蛋白与肌动蛋白混合以引发肌动蛋白相互作用。肌动蛋白活化幂的动力学动力学将通过瞬时分辨荧光法对继电器螺旋的构象变化进行监测。我们将回答以下问题：力产生的时间是什么，与核苷酸结合以及水解释放产物有何关系？目标2。肌球蛋白ATPase肌动蛋白激活的机理。将使用脉冲电子顺磁共振和瞬时时间分辨荧光法研究中继螺旋构象及其在功能性肌球蛋白突变体中的瞬时结构动力学。我们将回答两个问题：继电器循环是否是肌动蛋白肌动蛋白激活的调节元素？这种激活的机制是什么？这项研究对于理解肌肉功能和肌肉故障的机制至关重要。这些机制的知识将允许对肌肉故障治疗的合理设计，并更好地理解肌肉收缩的机制。公共卫生相关性：该项目的最终目标是了解肌肉中的力量是如何产生的。在拟议的研究中，我们将研究肌动蛋白是如何通过肌动蛋白激活力生产的分子运动的。为了达到目标，我们将结合分子生物学，生物化学和生物物理光谱法。这项研究对于理解肌肉功能和肌肉故障的机制至关重要。了解这种机制将允许对肌肉故障治疗的合理设计，并更好地了解肌肉收缩的机制。