Myosin structural regions that determine muscle shorten*

描述（由申请人提供）：肌球蛋白将ATP水解的化学能转化为力和运动的动力学和结构机制尚不清楚。普遍接受的肌凝蛋白过桥理论认为Pi释放与产生力量冲程有关，但与ADP释放率相关的步骤被认为是对卸载缩短速度的限速。然而，最近来自肌凝蛋白分子研究的证据表明，ADP的释放并不是影响未卸载肌凝蛋白缩短速度的唯一步骤。此外，果蝇肌球蛋白和飞行肌纤维的动力学研究表明，快速的果蝇肌球蛋白可能受到与Pi释放相关的步骤而不是ADP释放速率的限制。因此，需要验证的动力学假设是，快速肌凝蛋白的卸载速度受到与Pi释放相关的步骤的限制，而缓慢肌凝蛋白的速度主要受到ADP释放速率的限制。具体目的：(1)通过在体外滑动丝实验中改变ATP、Pi和ADP水平，在分子水平上验证我们的动力学假设。我们将比较两种速度非常快的成年果蝇同型体和两种速度缓慢的胚胎果蝇同型体对速度的影响。(2)通过改变三磷酸腺苷（ATP）、磷酸腺苷（Pi）和磷酸腺苷（ADP）在转基因表达四种肌球蛋白异构体的TDT（跳）皮肤洗浴液中的水平，测试Pi释放是否在纤维水平上限制了卸载速度。(3)确定哪个结构区域决定肌球蛋白同工型的缩短速度。我们的结构假设是肌凝蛋白转换区主要负责设置卸载速度的差异。我们将通过在两个肌凝蛋白嵌合体上进行与目标1和目标2中描述的相同的分子和纤维实验来验证这一假设，这些嵌合体以前是通过在非常快和非常慢的肌凝蛋白异构体之间交换转换区域而制成的。意义：根据动力学假设的有效性，我们将确定哪个区域影响ADP释放或哪个区域影响Pi释放。后者将是非常重要的，因为肌球蛋白的区域设定Pi释放率尚未确定。无论哪种情况，关于转换区功能的信息都将具有很高的信息量，因为转换区是导致家族性肥厚性心肌病（FHC）突变的热点。FHC是一种遗传性遗传病，是年轻人猝死的主要原因。

DESCRIPTION (provided by applicant): The kinetic and structural mechanisms by which myosin converts the chemical energy of ATP hydrolysis to force and motion are far from being understood. Generally accepted myosin cross-bridge theories have Pi release associated with the force producing power stroke, but steps associated with ADP release rate are thought to be rate limiting for unloaded shortening velocity. However, recent evidence from myosin molecular studies suggests that ADP release is not the only step of the cycle that can influence unloaded myosin shortening velocity. Further, kinetic studies of Drosophila myosins and flight muscle fibers suggest that fast Drosophila myosins may be limited by steps associated with Pi release rather than ADP release rate. Therefore, the KINETIC HYPOTHESIS to be tested is that the unloaded velocity of very fast myosins is limited by steps associated with Pi release while slow myosin velocities are limited primarily by ADP release rate. SPECIFIC AIMS: (1) Test our kinetic hypothesis at the molecular level by varying ATP, Pi and ADP levels in the in vitro sliding filament assay. We will contrast the effect on velocity for two very fast adult Drosophila isoforms compared with two slow embryonic isoforms. (2) Test if Pi release limits unloaded velocity at the fiber level by varying ATP, Pi and ADP levels in the bathing solution of skinned TDT (jump) muscle transgenically expressing the four myosin isoforms. (3) Determine which structural region sets shortening velocity of myosin isoforms. Our STRUCTURAL HYPOTHESIS is that the myosin converter region is primarily responsible for setting differences in unloaded velocity. We will test this hypothesis by performing the same molecular and fiber experiments as described in Aims 1 and 2 on two myosin chimeras previously made by exchanging converter regions between a very fast and a very slow myosin isoform. Significance: Depending on the validity of our kinetic hypothesis, we will either be determining which region influences ADP release or which region influences Pi release. The latter would be highly significant as regions of myosin that set Pi release rate have not been identified. In either case, information on the function of the converter region will be highly informative as the converter is a hotspot for mutations that lead to familial hypertrophic cardiomyopathy (FHC). FHC is an inherited genetic disease that is a major cause of sudden death among young adults.