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: 7157560
• 负责人: DOUGLAS M SWANK
• 金额: $ 7.81万
• 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/7157560
• 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（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远未被理解。普遍接受的肌球蛋白跨桥理论具有与产生动力冲程的力相关的Pi释放，但是与ADP释放速率相关的步骤被认为是无负荷缩短速度的速率限制。然而，肌球蛋白分子研究的最新证据表明，ADP的释放不是唯一的步骤，可以影响卸载肌球蛋白缩短速度的周期。此外，果蝇肌球蛋白和飞行肌纤维的动力学研究表明，快速果蝇肌球蛋白可能是有限的步骤与Pi的释放，而不是ADP的释放速率。因此，待检验的动力学假设是，非常快的肌球蛋白的无负荷速度受到与Pi释放相关的步骤的限制，而慢的肌球蛋白速度主要受到ADP释放速率的限制。具体目的：（1）在体外滑丝试验中，通过改变ATP、Pi和ADP水平，在分子水平上检验我们的动力学假设。我们将比较两个非常快的成年果蝇亚型与两个缓慢的胚胎亚型的速度的影响。(2)通过改变转基因表达四种肌球蛋白同种型的带皮TDT（跳跃）肌肉的浴液中的ATP、Pi和ADP水平，测试Pi释放是否限制了纤维水平的无负荷速度。(3)确定哪个结构区域决定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无负荷速度的差异。我们将通过对两个肌球蛋白嵌合体进行与目标1和2中所述相同的分子和纤维实验来检验这一假设，这两个肌球蛋白嵌合体是通过在非常快的肌球蛋白同种型和非常慢的肌球蛋白同种型之间交换转换器区域而制成的。重要性：根据我们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，因为尚未确定设定Pi释放速率的肌球蛋白区域。在任何一种情况下，关于转换区功能的信息将是高度信息化的，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。

项目成果

An alternative domain near the ATP binding pocket of Drosophila myosin affects muscle fiber kinetics.

• DOI: 10.1529/biophysj.105.075184
• 发表时间: 2006-04
• 期刊: Biophysical journal
• 影响因子: 3.4
• 作者: D. Swank;Joan M. Braddock;Waylon Brown;Heather Lesage;S. Bernstein;D. Maughan