MECHANICS OF KINESIN--A MICROTUBULE BASED MOTOR PROTEIN

驱动蛋白的机制——一种基于微管的运动蛋白

• 批准号: 2080145
• 负责人: Jonathon Howard
• 金额: $ 19.35万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-06-30 至 2000-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2080145
• 关键词: affinity chromatography; animal tissue; cell motility; dynein ATPase; gel filtration chromatography; high performance liquid chromatography; kinesin; membrane activity; membrane channels; microtubules; myosins; protein structure function; site directed mutagenesis; video microscopy

The long-term objective of the proposed studies is to understand how motor proteins work. These enzymes, which include myosin from muscle, dynein from cilia and flagella, and kinesin from eukaryotic cells in general, convert the chemical energy contained in the gamma phosphate bond of ATP into mechanical work used to power intracellular transport. Several molecular models for force generation, most notably the crossbridge-cycle model, have been formulated based on ATPase assays, mechanical recordings from muscle, and structural studies. The strategy of this proposal is to directly test these models by using recently-developed, highly-sensitive physical techniques to measure force and displacement at the single-molecule level. Single kinesin molecules will be placed under various known loads by challenging each one to pull on a microtubule attached to a minute calibrated flexible glass fiber. The motion of the motor will be measured by imaging the tip of the fiber onto a photodiode detector with subnanometer precision and submillisecond time resolution. The mechanical performance of individual motors will be tested under a wide range of loads, ATP concentrations, and orientations. The mechanical components of the motor, including the elastic element posited by the crossbridge cycle model, will be characterized physically; and the change in strain in this elastic element, the powerstroke, will be measured. A crucial prediction of the crossbridge cycle model will be tested by comparing the single-motor force with the product of the elastic element's stiffness and the powerstroke distance. Using site-directed mutagenesis we hope to identify which amino acids form the various mechanical components, and propose to determine the role of kinesin's two heads. Lastly, by combining biochemical techniques with the newly developed optical tweezer technology, we propose to measure the distance moved per ATP hydrolyzed: the simplest version of the model predicts that this distance should equal the 8-nm step size. Because of the structural and biochemical similarities between kinesin, myosin, and dynein, the elucidation of the molecular events underlying energy transduction by kinesin should significantly increase the understanding of cellular motility in general. It is hoped that this understanding may lead to more rational treatments of muscle disorders such as heart disease, or to better methods of selectively interfering with pathological cellular movements such as the invasion and proliferation of tumor cells, and the transport of viruses between the cell membrane and the nucleus.

拟议研究的长期目标是了解运动方式 蛋白质起作用。这些酶，包括肌肉，动力蛋白 从纤毛和鞭毛，以及一般来自真核细胞的动力素， 转换ATPγ磷酸键中包含的化学能 进入用于为细胞内运输供电的机械工作。一些 力产生的分子模型，最著名的是Crossbridge-cycle 模型已根据ATPase分析，机械记录制定 来自肌肉和结构研究。 该建议的策略是通过使用 最近开发的，高敏感的物理技术来测量力 和单分子水平的位移。单驱蛋白分子 通过挑战每个人的拉力，将被放置在各种已知负载下 在连接到微小校准的柔性玻璃纤维上的微管上。这 电动机的运动将通过对纤维的尖端进行成像来测量 具有亚纳光度精度和亚数的光电二极管检测器 解决。单个电动机的机械性能将进行测试 在各种负载，ATP浓度和方向下。这 电动机的机械组件，包括放置的弹性元件 通过Crossbridge循环模型，将在物理上进行表征。和 这种弹性元素的应变变化，力量将是 测量。 Crossbridge循环模型的关键预测将是 通过将单运动力与弹性的乘积进行比较来测试 元素的刚度和力量距离。使用站点定向 诱变我们希望确定哪种氨基酸形成了各种 机械组件，并提出确定驱动蛋白的作用 头。最后，通过将生化技术与新技术相结合 开发的光学镊子技术，我们建议测量距离 移动的每个ATP水解：模型的最简单版本预测 此距离应等于8 nm步长。 由于驱动蛋白之间的结构和生化相似性， 肌球蛋白和Dynein，阐明了分子事件的基础事件 驱动蛋白的能量转导应显着增加 总体上了解细胞运动。希望这个 理解可能会导致对肌肉障碍的更多理性治疗 例如心脏病或更好地干扰的更好方法 具有病理细胞运动，例如入侵和 肿瘤细胞的增殖，并在 细胞膜和细胞核。