权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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(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。在肌球蛋白II中，肌动蛋白结合启动了PowerStroke，这是一个单一的力产生事件，当肌球蛋白改变其构象并推动肌肉中相对于粗丝的肌动蛋白细丝或在细胞内沿着肌动蛋白细胞骨架运输货物时。Acto-myosin相互作用调节肌球蛋白ATPase活性，提高ATP水解物的释放速率。本项目的目标是确定肌球蛋白ATPase肌动蛋白激活的机制。我们假设，PowerStroke是由肌动蛋白通过调节肌球蛋白力量生成区的继电器环-继电器螺旋相互作用来调节的。肌动蛋白结合改变了PowerStroke前阶段的继电器环构象，破坏了继电器螺旋的稳定性，从而诱发了PowerStroke。我们将使用新开发的方法来测量肌球蛋白-ATP和肌球蛋白-肌动蛋白相互作用过程中继发螺旋的构象，该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时时间分辨荧光技术，这是两种互补的高分辨率光谱技术。如前所述，这些突变和标记不会影响肌球蛋白的功能。我们将在中继环中引入功能突变来扰乱肌动蛋白的激活，并详细研究激活机制，分析肌球蛋白在肌动蛋白激活过程中的构象和这种构象变化的时间。目的1.研究肌球蛋白肌动蛋白激活力产生力的技术进展。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中继螺旋构象变化的动力学。我们将现有的瞬变时间分辨荧光仪升级为3个注射器/2个混合器停流仪。首先，肌球蛋白将在PowerStroke前的短暂状态下通过与ATP快速混合而制备。然后，我们将准备好的肌球蛋白与肌动蛋白快速混合，以启动肌球蛋白与肌球蛋白的相互作用。肌动蛋白激活PowerStroke的动力学将通过瞬时时间分辨荧光法通过继电螺旋的构象变化来监测。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释放的产物有什么关系？目的2.肌球蛋白ATPase肌动蛋白激活的机制。利用脉冲电子顺磁共振和瞬时时间分辨荧光技术研究功能性肌球蛋白突变体的继发螺旋构象及其瞬时结构动力学。我们将回答两个问题：中继环是肌球蛋白肌动蛋白激活的调节元件吗？这种激活的机制是什么？本研究对于从亚分子水平上理解肌肉功能和肌肉功能障碍的机制具有重要意义。对这些机制的了解将有助于合理设计肌肉功能障碍的治疗方法，以及更好地了解肌肉收缩的机制。与公共卫生相关：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，我们将研究肌球蛋白这一分子马达是如何被肌动蛋白激活以产生力量的。为了达到这个目标，我们将把分子生物学、生物化学和生物物理光谱学结合起来。本研究对于从亚分子水平上理解肌肉功能和肌肉功能障碍的机制具有重要意义。对这一机制的了解将有助于合理设计肌肉功能障碍的治疗方法，以及更好地了解肌肉收缩的机制。