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马达如何 蛋白质起作用。这些酶，包括来自肌肉的肌球蛋白，动力蛋白 来自纤毛和鞭毛，以及来自真核细胞的动蛋白， 转换三磷酸腺苷的伽马磷酸键中所含的化学能 转化为用于细胞内运输的机械功。几个 力产生的分子模型，最著名的是交叉桥循环 模型，都是基于ATPase分析，机械记录 来自肌肉和结构研究。 该提案的策略是通过使用以下方法直接测试这些模型 最近发展起来的高灵敏度的测量力的物理技术 和单分子水平上的位移。单个动蛋白分子 会通过挑战每一个人来承受各种已知的负荷 在连接到微小校准的柔性玻璃纤维上的微管上。这个 电机的运动将通过将光纤的尖端成像到 一种亚纳米精度、亚毫秒时间的光电二极管探测器 决议。将测试单个电机的机械性能 在大范围的负荷、ATP浓度和方向下。这个 电机的机械部件，包括放置的弹性元件 通过跨桥循环模型，将被物理地表征；以及 这个弹性元件PowerStroke中的应变变化将是 量过了。跨桥周期模型的一个关键预测将是 通过比较单电机的力和弹性的乘积进行测试 元素的刚度和动力冲程。使用站点定向 突变我们希望确定哪些氨基酸形成了不同的 机械部件，并建议确定Kinesin的两个 头朝上。最后，通过将生化技术与新的 发展了光钳技术，我们提出了测量距离的方法 按ATP水解物移动：模型的最简单版本预测 此距离应等于8纳米步长。 由于动蛋白在结构和生化上的相似性， 肌球蛋白和动力蛋白，阐明潜在的分子事件 激动素的能量转导应该会显著增加 对细胞运动性的一般理解。希望这是一次 了解这一点可能会导致对肌肉疾病的更合理的治疗 例如心脏病，或更好的选择性干预方法 有病理性的细胞运动，如入侵和 肿瘤细胞的增殖，以及病毒在 细胞膜和细胞核。