Myosin structural regions that determine muscle shorten*

决定肌肉缩短的肌球蛋白结构区域*

• 批准号: 7141262
• 负责人: DOUGLAS M SWANK
• 金额: $ 3.12万
• 依托单位: RENSSELAER POLYTECHNIC INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-12 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7141262
• 关键词: Drosophilidae; adenosine diphosphate; adenosine triphosphate; bioenergetics; biomechanics; biophysics; embryo /fetus protein; fiber cell; fluorescence microscopy; muscle tension; myosins; phosphates; protein isoforms; protein structure function; protein transport; video microscopy

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.

描述(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还远未被了解。普遍接受的肌球蛋白跨桥理论认为PI释放与产生功率卒中的力有关，但与ADP释放速率相关的步骤被认为是对无负荷缩短速度的速率限制。然而，最近来自肌球蛋白分子研究的证据表明，ADP的释放并不是影响卸载的肌球蛋白缩短速度的唯一步骤。此外，对果蝇肌球蛋白和飞行肌肉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释放相关的步骤的限制，而不是ADP释放速度的限制。因此，需要检验的动力学假说是，非常快的肌球蛋白的卸载速度受到与PI释放相关的步骤的限制，而缓慢的肌球蛋白速度主要受ADP释放速度的限制。具体目的：(1)通过在体外滑丝实验中改变ATP、PI和ADP的水平，在分子水平上验证我们的动力学假说。我们将对比两个非常快的成年果蝇亚型和两个缓慢的胚胎亚型对速度的影响。(2)通过改变转基因表达四种肌球蛋白异构体的皮肤TDT(JUMP)肌肉浴液中的ATP、PI和ADP水平，测试PI释放是否在纤维水平限制了卸载速度。(3)确定哪个结构区域决定了肌球蛋白亚型的缩短速度。我们的结构假设是，肌球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球蛋白嵌合体上执行与目标1和2中描述的相同的分子和纤维实验来验证这一假设，这两个嵌合体是通过在非常快的和非常慢的肌球蛋白亚型之间交换转换区域而形成的。意义：根据我们动力学假说的有效性，我们将确定哪个区域影响ADP的释放，哪个区域影响PI的释放。后者非常重要，因为肌球蛋白决定PI释放率的区域尚未确定。在任何一种情况下，关于转换区功能的信息都将是非常有用的，因为转换区是导致家族性肥厚型心肌病(FHC)的突变的热点。FHC是一种遗传性疾病，是年轻人猝死的主要原因。