Myosin structural and kinetic mechanisms that differentiate fast and slow muscle

描述(由申请人提供)：肌球蛋白和其他马达蛋白将ATP水解的化学能转化为力和运动的动力学和结构机制还远未被了解。在普遍接受的肌球蛋白跨桥功能理论中，PI释放与力量产生的力量卒中有关，但与镁ADP释放速率相关的步骤被认为是对无负荷肌肉缩短速度和振荡功产生的速率限制。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是由PI释放速率而不是镁ADP释放速率设置的(Swank等人，2006年)。因此，我们将检验我们的动力学假说，即非常快的肌球蛋白的卸载速度和振荡功产生受到与PI释放相关的步骤的限制，而较慢的肌球蛋白受到与镁ADP释放速率相关的步骤的限制。具体目标：(1)通过改变间接飞行肌肉(IFM)中的镁ATP、PI和镁ADP水平来测试我们关于振荡功产生的动力学假说，以确定包括限速步骤在内的关键跨桥速率常数。(2)通过改变转基因表达相同四种肌球蛋白亚型的果蝇跳肌浴液中的镁ATP、PI和镁ADP水平，测试PI或ADP释放是否在纤维水平限制卸载速度。我们的动力学假说也将通过肌动蛋白滑动细丝分析在分子水平上得到验证。(3)验证我们的结构假说，即肌球蛋白转化器是决定跨桥速率常数值的主要区域，而跨桥速率常数值对于设定肌球蛋白亚型的缩短速度至关重要。我们将通过对肌球蛋白嵌合体进行与目标1和2中描述的相同的分子和纤维实验来验证这一假设，该嵌合体是通过用果蝇转换区的其他4个天然版本替换IFM肌球蛋白转换器而制成的。(4)通过替换降低IFM肌球蛋白异构体疏水性的氨基酸来检验我们的机制假说，即转化器中的疏水性程度对其功能至关重要。意义：我们将确定转换区域如何影响镁ADP释放和/或PI释放。这将是非常重要的，因为人们对马达蛋白决定PI和镁ADP释放率的结构机制知之甚少。关于转化器设定速度的机制的细节将有助于检验最近的假设，即至少8种不同的转化器突变如何导致家族性肥厚型心肌病(FHC)或扩张型心肌病(DCM)。FHC是一种遗传性疾病，是年轻人猝死的主要原因。

DESCRIPTION (provided by applicant): 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 cardiomyopathy (DCM). FHC is an inherited genetic disease that is a major cause of sudden death among young adults.