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: 6129909
• 负责人: Linda Wordeman
• 金额: $ 29.49万
• 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 2001-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6129909
• 关键词: X ray crystallography; animal tissue; cell motility; dynein ATPase; elasticity; fluorescence microscopy; genetic manipulation; hydrolysis; intracellular transport; kinesin; membrane activity; membrane channels; microtubules; myosins; plasmids; protein engineering; protein purification; protein structure function; video microscopy

Description: (Verbatim from the applicant's abstract.) 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 derived from hydrolysis of the gamma phosphate bond of ATP into mechanical work used to power intracellular transport. The strategy of this proposal, which focuses on the microtubule-based motor kinesin, is to combine high-sensitivity single-molecule techniques with biochemical and protein engineering techniques in order to identify the moving parts of the motor-the springs, levers, and axles-and to understand how their coordinated motion is coupled to the hydrolysis of ATP. Kinesin is a processive motor capable of making many steps along a microtubule without dissociating. We will test whether processivity is due to mechanical coordination between kinesin's two motor domains by measuring how force effects the dissociation of individual heads from the microtubule. Putative elastic elements will be localized, and 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. Based on the approximate two-fold symmetry of dimeric kinesin when both its heads are in the same nucleotide state, we hypothesize that the power stroke is associated with a rotation of one head with respect to the other: we will use single-molecule fluorescence microscopy to visualize this rotation. To determine how tight is the coupling between chemical and mechanical steps, we will measure the effect of load on the ATP hydrolysis rate. These results will be incorporated into a kinetic model that will relate the speed and processivity of the motor to the load it carries and the ATP concentration. 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.

描述：(逐字摘自申请人的摘要。)长期目标 建议的研究之一是了解马达蛋白是如何工作的。这些 酶，包括来自肌肉的肌球蛋白，来自纤毛和鞭毛的动力蛋白，以及 动蛋白一般从真核细胞中转化而来的化学能 从三磷酸腺苷的伽马磷酸键水解为机械功，用于 为细胞内运输提供动力。 这一方案的战略重点是基于微管的电机 动蛋白，是将高灵敏度的单分子技术与 生化和蛋白质工程技术，以识别移动 发动机的部件-弹簧、杠杆和车轴-并了解它们的 协调运动与三磷酸腺苷的水解相结合。激动素是一个过程 能够沿着微管走很多步而不会分离的马达。我们 将测试处理性是否是由于 通过测量力如何影响运动蛋白的解离 从微管中分离出单个头部。假定的弹性元件将是 本地化，跨桥循环模型的关键预测将是 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。基于近似值 当两个头在同一位置时二聚体动蛋白的双重对称性 核苷酸状态时，我们假设力量卒中与 一个头相对于另一个头的旋转：我们将使用单分子 用荧光显微镜观察这种旋转。要确定有多紧， 化学步骤和机械步骤之间的耦合，我们将测量其效果 负荷对ATP水解率的影响。这些结果将被合并到一个 动力学模型将把电机的速度和加工能力与 它所携带的负荷和ATP浓度。 因为肌动蛋白，肌球蛋白， 和动力蛋白，阐明了能量背后的分子事件 通过激动素的转导应该会显著增加对 一般来说，细胞的运动性。希望这一谅解可能导致 更合理的治疗肌肉疾病，如心脏病，或 选择性干扰病理性细胞运动的更好方法 如肿瘤细胞的侵袭和增殖，以及肿瘤的运输 位于细胞膜和细胞核之间的病毒。