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，以及来自一般真核细胞的驱动蛋白， 转换 ATP 的 γ 磷酸键中包含的化学能 转化为用于为细胞内运输提供动力的机械功。一些 力产生的分子模型，尤其是跨桥循环 模型，是根据 ATP 酶测定、机械记录制定的 来自肌肉和结构研究。 该提案的策略是使用以下方法直接测试这些模型 最近开发的高灵敏度物理技术来测量力 和单分子水平的位移。单驱动蛋白分子 通过挑战每个人拉动，将被置于各种已知负载下 在连接到微小校准柔性玻璃纤维的微管上。这 电机的运动将通过将光纤尖端成像到 具有亚纳米精度和亚毫秒时间的光电二极管探测器 解决。将测试单个电机的机械性能 在各种负载、ATP 浓度和方向下。这 电机的机械部件，包括设置的弹性元件 通过跨桥循环模型，将进行物理表征；和 该弹性元件的应变变化（动力冲程）将是 测量。跨桥循环模型的一个关键预测是 通过比较单电机力与弹性力的乘积进行测试 元件的刚度和动力冲程距离。使用站点定向 诱变我们希望确定哪些氨基酸形成了不同的 机械部件，并建议确定驱动蛋白的两个作用 头。最后，通过将生化技术与新的技术相结合 开发光镊技术，我们建议测量距离 每 ATP 水解移动：该模型的最简单版本预测 该距离应等于 8 nm 步长。 由于驱动蛋白之间的结构和生化相似性， 肌球蛋白和动力蛋白，阐明其背后的分子事件 驱动蛋白的能量转换应显着增加 对细胞运动的一般理解。希望这 了解可能会导致更合理地治疗肌肉疾病 例如心脏病，或者更好的选择性干扰方法 病理性细胞运动，例如侵袭和 肿瘤细胞的增殖和病毒在肿瘤细胞之间的运输 细胞膜和细胞核。