Mechanics of Kinesin: a Microtubule-Based Motor Protein

这些研究的长期目标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来自真核细胞的驱动蛋白，通常将ATP的γ磷酸键水解产生的化学能转化为用于为细胞内运输提供动力的机械功。该提案的战略，重点是基于微管的运动驱动蛋白，是联合收割机高灵敏度的单分子技术与生物化学和蛋白质工程技术，以联合收割机高灵敏度的单分子与生物化学和蛋白质工程技术，以确定运动的部分-弹簧，水平和轴-并了解他们的协调运动是如何耦合到ATP的水解。驱动蛋白是一种能够沿着微管沿着多个步骤而不解离的进行性马达。我们将测试是否proventivity是由于驱动蛋白的两个电机域之间的机械协调，通过测量力如何影响单个头从微管的解离。假定的弹性元件将被本地化，并且通过将单个电机力与弹性元件的刚度和动力行程距离的乘积进行比较，来测试跨桥循环模型的关键预测。我们将直接确定是否结合核苷酸的变化改变驱动蛋白的两个头部的流动性，通过测量在不同的核苷酸条件下的驱动蛋白的扭转刚度。基于二聚体驱动蛋白在其两个头部处于相同核苷酸条件下的近似双重对称性。基于当二聚体驱动蛋白的两个头部处于相同核苷酸状态时其近似双重对称性，我们假设动力冲程与一个头部相对于另一个头部的旋转相关：我们将使用单分子荧光显微镜来可视化该旋转。为了确定化学和机械步骤之间的耦合有多紧密，我们将测量负载对ATP水解速率的影响。我们将建立一个动力学模型来综合这些力学结果和驱动蛋白的生化反应。由于驱动蛋白，肌球蛋白和动力蛋白之间的结构和生物化学的相似性，阐明驱动蛋白的能量转导的分子事件应显着增加一般的细胞运动的理解。希望这种理解可能会导致更合理的治疗肌肉疾病，如心脏病，或选择性干扰病理细胞运动的更好方法，如肿瘤细胞的入侵和增殖，以及细胞膜和细胞核之间的病毒运输。

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 combine high-sensitivity single-molecule with biochemical and protein engineering techniques in order to identify the moving parts-the springs, levels, 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 procesivity is due to mechanical coordination between kinesin's tow 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. We will directly determine whether changes in bound nucleotide alter the mobility of kinesin's two heads, by measuring the torsional stiffness of kinesin under different nucleotide conditions. Based on the approximately two-fold symmetry of dimeric kinesin when both its heads are in the same nucleotide conditions. 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. A kinetic model will be developed to synthesize these mechanical results with biochemical of kinesin. 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.