Myosin structural and kinetic mechanisms that differentiate fast and slow muscle

D.M.斯旺克项目摘要/摘要 肌球蛋白和其他马达蛋白转化成 人们对三磷酸腺苷的化学能的作用和运动还知之甚少。一般说来 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，但与镁ADP释放率相关的步骤被认为是对卸载的速率限制 肌肉缩短速度和振荡功产生。然而，最近的证据表明， 镁ADP的释放并不是影响肌肉缩短速度的唯一步骤，我们的 最近的数据表明，果蝇肌球蛋白产生振荡功的最佳频率 由PI释放率而不是镁ADP释放率设定(Swank等人，2006年)。因此，我们将 测试我们的动力学假设，即空载速度和振荡功产生的速度非常快 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 与镁ADP释放率相关。 具体目标：(1)通过改变镁ATP，PI来测试我们的振荡功生产的动力学假说 转基因表达四种果蝇肌球蛋白的间接飞行肌肉(IFM)中的镁ADP水平 速度变化9倍的异构体，以确定关键的跨桥速率常数，包括 速率限制步骤。(2)测试PI或ADP释放是否在纤维水平限制卸载速度 转基因果蝇跳肌浴液中镁ATP、PI和镁ADP含量的变化 表达相同的四种肌球蛋白亚型。我们的动力学假说也将在分子水平上得到验证 水平使用肌动蛋白滑动细丝分析。(3)测试我们的结构假设，即肌球蛋白 转换器是负责确定跨桥速率常数值的主要区域，这些值对 设定肌球蛋白亚型的缩短速度。我们将通过执行相同的操作来验证这一假设 目标1和目标2中描述的对肌球蛋白嵌合体通过替换 IFM肌球蛋白转换区与果蝇转换区的其他4个天然版本。(4)测试 我们的机械假设是，转化器中的疏水性程度对其 通过取代降低IFM肌球蛋白异构体疏水性的氨基酸发挥作用。 意义：我们将确定转换区域如何影响镁ADP释放和/或PI 放手。这将是非常重要的，因为人们对其结构机制知之甚少 马达蛋白决定PI和镁ADP的释放率。有关转换器的设置机制的详细信息 Velity将帮助测试最近关于转换器中至少8种不同突变的假设 引起家族性肥厚型心肌病(FHC)或扩张型心肌病(DCM)。FHC是一种 遗传性遗传病，是年轻人猝死的主要原因。斯万克，D.M.项目叙事 通过对分子马达肌球蛋白的力学和生物化学的研究，发现其分子马达肌球蛋白 增强心肌收缩，我们将学习肌球蛋白突变是如何导致两种 心脏病类型、家族性肥厚型心肌病(FHC)和扩张型 心肌病(DCM)。FHC是运动员猝死的主要原因， 年轻人(Morita等人，2005年)。相比之下，扩张型心肌病通过 心脏肌肉质量的丧失和心脏体积的有害增加。

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.