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Myosin structural and kinetic mechanisms that differentiate fast and slow muscle

区分快肌和慢肌的肌球蛋白结构和动力学机制

基本信息

批准号：
8318002
负责人：
DOUGLAS M SWANK
金额：
$ 28.34万
依托单位：
RENSSELAER POLYTECHNIC INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-09-15 至 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8318002
关键词：
ATP Hydrolysis Acceleration Actins Actomyosin Affinity Amino Acids Animal Model Area Bathing Biochemistry Biological Assay Cardiac Volume Chemicals Chimera organism Data Dilated Cardiomyopathy Drosophila genus Embryo Familial Hypertrophic Cardiomyopathy Fiber Filament Frequencies Generations Genes Heart Heart Diseases Heart failure Hereditary Disease Human Hydrophobic Interactions Hydrophobicity Inherited Kinetics Learning Measures Mechanics MgADP MgATP Microfilaments Modeling Molecular Molecular Motors Motion Motor Muscle Muscle Contraction Muscle Fibers Mutate Mutation Myocardium Myosin ATPase N-terminal Power stroke Production Property Protein Isoforms Proteins RNA Splicing Set protein Skin Slide Solutions Speed Stress Sudden Death Testing Work abstracting alpha helix arm base cell motility inorganic phosphate muscle form research study response skeletal theories young adult

项目摘要

Swank, D.M. Project Summary/Abstract The kinetic and structural mechanisms by which myosin and other motor proteins convert the chemical energy of ATP hydrolysis into force and motion are far from being understood. In generally accepted theories of myosin cross-bridge function, Pi release is associated with the force-producing power stroke, but steps associated with MgADP release rate are thought to be rate limiting for unloaded muscle shortening velocity and oscillatory work production. However, recent evidence suggests that MgADP release is not the only step of the cycle that influences muscle shortening velocity, and our recent data suggest the optimal frequency of oscillatory work production by very fast Drosophila myosin is set by the Pi release rate rather than the MgADP release rate (Swank et al., 2006). Therefore, we will test our KINETIC HYPOTHESIS that unloaded velocity and oscillatory work production by very fast myosins are limited by steps associated with Pi release while slower myosins are limited by steps associated with MgADP release rate. SPECIFIC AIMS: (1) Test our kinetic hypothesis for oscillatory work production by varying MgATP, Pi and MgADP levels in indirect flight muscle (IFM) transgenically expressing four Drosophila myosin isoforms, which vary 9-fold in velocity, to determine critical cross-bridge rate constants including the rate limiting step. (2) Test if Pi or ADP release limits unloaded velocity at the fiber level by varying MgATP, Pi and MgADP levels in the bathing solution of skinned Drosophila jump muscle transgenically expressing the same four myosin isoforms. Our kinetic hypothesis will also be tested at the molecular level using the actin sliding filament assay. (3) Test our STRUCTURAL HYPOTHESIS that the myosin converter is the primary region responsible for determining cross-bridge rate constant values critical for setting the shortening velocity of myosin isoforms. We will test this hypothesis by performing the same molecular and fiber experiments as described in Aims 1 and 2 on myosin chimeras made by replacing the IFM myosin converter with the other 4 native versions of the Drosophila converter region. (4) Test our MECHANISTIC HYPOTHESIS that the degree of hydrophobicity in the converter is critical to its function by substituting amino acids that decrease the IFM myosin isoform's hydrophobicity. SIGNIFICANCE: We will determine how the converter region influences MgADP release and/or Pi release. This will be highly significant as very little is known about the structural mechanisms by which motor proteins set Pi and MgADP release rates. Details about the converter's mechanism for setting velocity will help test recent hypotheses regarding how at least 8 different mutations in the converter cause either familial hypertrophic cardiomyopathy (FHC) or dilated cardiaomyopathy (DCM). FHC is an inherited genetic disease that is a major cause of sudden death among young adults. Swank, D.M. Project Narrative By studying the mechanics and biochemistry of the molecular motor myosin, which powers heart muscle contraction, we will learn how mutations in myosin cause two types of heart disease, familial hypertrophic cardiomyopathy (FHC) and dilated cardiomyopathy (DCM). FHC is the leading cause of sudden death in athletes and young adults (Morita et al., 2005). In contrast, DCM results in heart failure through a loss of muscle mass from the heart and a detrimental increase in heart volume.

Swank，D. M.项目总结/摘要肌球蛋白和其他运动蛋白将肌球蛋白转化为肌球蛋白的动力学和结构机制， ATP水解成力和运动的化学能还远未被理解。以大致公认的肌球蛋白跨桥功能理论，Pi的释放与力的产生有关。动力冲程，但与MgADP释放速率相关的步骤被认为是无负荷的速率限制肌肉缩短速度和振荡功产生。然而，最近的证据表明， MgADP的释放并不是影响肌肉缩短速度的唯一步骤，我们的研究表明，最近的数据表明，非常快的果蝇肌球蛋白产生振荡功的最佳频率由Pi释放速率而不是MgADP释放速率设定（Swank等人，2006年）。所以我们会测试我们动力学假设，即空载速度和振荡功产生非常快肌球蛋白受与Pi释放相关的步骤限制，而较慢的肌球蛋白受步骤限制与MgADP释放速率相关。具体目的：（1）通过改变MgATP，Pi，转基因表达四种果蝇肌球蛋白的间接飞行肌（IFM）中的MgADP和MgADP水平异构体，其速度变化9倍，以确定临界跨桥速率常数，包括速率限制步骤(2)测试Pi或ADP释放是否通过改变转基因去皮果蝇跳跃肌细胞培养液中MgATP、Pi和MgADP含量的变化表达相同的四种肌球蛋白同种型。我们的动力学假设也将在分子水平上得到检验。水平使用肌动蛋白滑动丝测定。(3)验证我们的结构假设，肌球蛋白转换器是负责确定跨桥速率常数值的主要区域，设定肌球蛋白同种型的缩短速度。我们将通过执行相同的操作来验证此假设。如目的1和2中所述，对通过替换 IFM肌球蛋白转换器与果蝇转换器区的其他4个天然版本。(4)测试我们的机械假设，即转化器中的疏水程度对其通过取代降低IFM肌球蛋白同种型疏水性的氨基酸来发挥作用。意义：我们将确定转换区如何影响MgADP释放和/或Pi release.这将是非常重要的，因为很少有人知道的结构机制，马达蛋白设定Pi和MgADP释放速率。有关转换器设置机制的详细信息速度将有助于测试最近的假设，关于如何至少8个不同的突变，在转换器引起家族性肥厚型心肌病（FHC）或扩张型心肌病（DCM）。FHC是一个一种遗传性疾病，是年轻人猝死的主要原因。Swank，D. M.项目叙述通过研究分子运动肌球蛋白的力学和生物化学，为心肌收缩提供动力，我们将了解肌球蛋白的突变如何导致两种类型的心脏病，家族性肥厚型心肌病（FHC）和扩张型心肌病心肌病（DCM）。FHC是运动员猝死的主要原因，年轻的成年人（Morita等人，2005年）。相比之下，扩张型心肌病通过心肌缺血导致心力衰竭。心脏肌肉质量的损失和心脏体积的有害增加。