STRUCTURE AND FUNCTION OF DYNEIN MOLECULES

动力蛋白分子与纤毛和鞭毛微管相互作用 产生纤毛跳动，伴随着三磷酸腺苷的水解。一个 了解他们的行动模式可能有助于分析这些 临床疾病如不孕不育、肺气肿和囊性纤维化，并可能 关于有丝分裂纺锤体的力学和微管介导的 胚胎学过程中的形状变化。已经在这些方面开发了方法 从体外分离提纯动力蛋白的实验室 和内臂，并在原位和体外可视化这些分子 采用快速冷冻深刻蚀技术。长期目标是 以现在可能的方式理解动力蛋白的力生成循环 肌球蛋白循环。描述了五个具体目标。1.外臂 动力蛋白将被解离成亚单位，并通过 高效液相色谱仪。通过将深度蚀刻与 ATPase活性、微管结合能力、 单抗结合等，应该是有可能的 生成蛋白质结构域的功能图。2.外臂动力 将在装饰微管和响应ATP时进行研究，这是目标 分析其三磷酸腺苷和微管蛋白的定位和修饰 结合部位。这些研究可望补充以下方面的研究 解离的动力蛋白。3.手臂内侧动力蛋白的功能图将是 并将评估它们在纤毛力学中的作用。4. 来自不同来源的不寻常的动力蛋白或类似动力蛋白的蛋白质 研究建立同源性并寻找与 “标准的”动力系统。5.将研究可移动的洗涤剂提取细胞以 了解动力蛋白如何改变它们的构型，以及它们的核苷酸 在原位节拍周期中的磷酸盐缔合。

Dynein molecules interact with ciliary and flagellar microtubules to generate the ciliary beat, with the concomitant hydrolysis of ATP. An understanding of their mode of action may facilitate the analysis of such clinical disorders as infertility, emphysema, and cystic fibrosis, and may pertain to the mechanics of the mitotic spindle and to microtubule-mediated shape changes during embryology. Methods have been developed in these laboratories for the isolation and purification of dyneins from the outer and inner arms, and for visualizing these molecules in situ and in vitro using the quick-freeze deep-etch technique. The long-term objective is to understand the dynein force-generating cycle in the fashion now possible for the myosin cycle. Five specific aims are described. 1. Outer-arm dynein will be dissociated into subunits and fractionated by high-performance liquid chromatography. By combining deep-etching with studies on ATPase activity, microtubule-binding ability, monoclonal-antibody binding, and the like, it should be possible to generate a functional map of the protein's domains. 2. Outer-arm dynein will be studied as it decorates microtubules and responds to ATP, the goal being to analyze the location and the modification of its ATP and tubulin binding sites. These studies will hopefully complement the studies on dissociated dynein. 3. A functional map of the inner-arm dyneins will be generated, and their role in ciliary mechanics will be assessed. 4. Unusual dyneins or dynein-like proteins from various sources will be studied to establish homologies and look for functional similarities with "standard" dyneins. 5. Motile detergent-extracted cells will be studied to learn how dyneins change their configuration, and their nucleotide phosphate associations, during the in situ beat cycle.