Cryo Electron Microscopy of Acto-S1 ATPase Intermediates

Acto-S1 ATPase 中间体的冷冻电子显微镜

• 批准号: 7093508
• 负责人: HOWARD D. WHITE
• 金额: $ 34.05万
• 依托单位: EASTERN VIRGINIA MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7093508
• 关键词: Cryo; Electron; Microscopy; Acto; S1

DESCRIPTION (provided by applicant): There is now strong if not overwhelming evidence that myosin-based movement results largely from a change in lever arm angle while the motor domain is attached to actin, in what is called the tilting lever mechanism. What is now required is to understand this mechanism in considerably greater detail, especially the correlation between different biochemical and structural states, details of the movement mechanisms in different myosin types, and whether the tilting mechanism alone provides a complete explanation of movement. A major aspect of our work will be to use time-resolved electron cryo-microscopy, which allows specimens to be examined under conditions very close to native and with excellent time resolution. We will use new image processing methods and automated electron microscopy to obtain the very large amounts of data that are necessary for high resolution images. Our goal will be to achieve <1 nm resolution, which allows internal alpha-helices in molecules to be revealed. If this can be done, there is the exciting prospect of seeing the individual elements, several of which are alpha helices, and how they change in different biochemical states to produce movement. Most of our work will use myosin V. Its 'weakly' bound states with actin have a much higher affinity than those of myosin II, allowing study of the pre-power stroke conformation. Moreover, the large size and asymmetry (6 calmodulins in the regulatory domain) make myosin V especially suitable for electron microscopy. However, we will also explore the use of non-muscle forms of myosin II (NMIIA and NMIIB) that have very slow rates of product release in the presence of actin. Our work is directly relevant to a number of diseases involving myosins. Myosin V has been identified as being the gene responsible for the "dilute mutation" in mice, which has defective transport of pigment to hair follicles, and is also responsible for developmental and neurological disorders. Non muscle myosin II plays key roles in platelet activation and the regulation of blood clotting.

描述（由申请人提供）：现在有强有力的（如果不是压倒性的）证据表明，基于肌球蛋白的运动主要是由于杠杆臂角度的变化，而运动域连接到肌动蛋白，即所谓的倾斜杠杆机制。现在需要的是更详细地了解这种机制，特别是不同生化和结构状态之间的相关性，不同肌球蛋白类型的运动机制的细节，以及倾斜机制是否单独提供了运动的完整解释。我们工作的一个主要方面将是使用时间分辨电子冷冻显微镜，它允许标本进行检查的条件下非常接近本地和优良的时间分辨率。我们将使用新的图像处理方法和自动电子显微镜来获得高分辨率图像所需的大量数据。我们的目标是实现<1 nm的分辨率，这使得分子中的内部α螺旋得以揭示。如果这一点能够实现，那么我们就有了一个激动人心的前景，可以看到单个元素，其中有几个是α螺旋，以及它们如何在不同的生化状态下发生变化以产生运动。我们的大部分工作将使用肌球蛋白V，其与肌动蛋白的“弱”结合状态具有比肌球蛋白II高得多的亲和力，允许研究前电力中风构象。此外，大的尺寸和不对称性（6钙调素的监管结构域），使肌球蛋白V特别适合电子显微镜。然而，我们也将探索使用非肌肉形式的肌球蛋白II（NMIIA和NMIIB），在肌动蛋白的存在下，产品释放速率非常慢。我们的工作与许多涉及肌球蛋白的疾病直接相关。肌球蛋白V已被确定为负责小鼠中的“稀释突变”的基因，该基因具有向毛囊的色素运输缺陷，并且还负责发育和神经系统疾病。非肌肉肌球蛋白II在血小板活化和血液凝固的调节中起关键作用。