Mechanics of Kinesin: a Microtubule-Based Motor Protein

拟议研究的长期目标是了解运动蛋白是如何工作的。这些酶包括来自肌肉的肌凝蛋白，来自纤毛和鞭毛的动力蛋白，以及来自真核细胞的动力蛋白，它们将ATP的伽马磷酸键水解产生的化学能转化为机械功，为细胞内运输提供动力。该提案的策略是将高灵敏度单分子技术与生物化学和蛋白质工程技术相结合，以便将高灵敏度单分子技术与生物化学和蛋白质工程技术相结合，以识别运动部件-弹簧，水平和轴-并了解它们的协调运动如何与ATP的水解相结合。肌动蛋白是一种进程马达，能够沿着微管进行许多步而不解离。我们将通过测量力如何影响单个头部与微管的分离来测试加工性是否由于驱动蛋白两个运动域之间的机械协调。假定的弹性元件将进行局部化，并通过将单电机力与弹性元件刚度和动力行程距离的乘积进行比较，来验证跨桥周期模型的关键预测。我们将通过测量不同核苷酸条件下酪蛋白的扭转刚度，直接确定结合核苷酸的变化是否会改变酪蛋白两个头部的流动性。基于二聚体动力蛋白的近似双重对称性，当它的两个头在相同的核苷酸条件下。基于二聚体驱动蛋白的近似双重对称性，当其两个头部处于相同的核苷酸状态时，我们假设功率冲程与一个头部相对于另一个头部的旋转有关：我们将使用单分子荧光显微镜来可视化这种旋转。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。将建立一个动力学模型，将这些力学结果与酶的生化反应综合起来。由于运动蛋白、肌凝蛋白和动力蛋白之间的结构和生化相似性，阐明运动蛋白能量转导的分子事件将显著增加对细胞运动的理解。人们希望，这种认识可能会导致更合理的治疗肌肉疾病，如心脏病，或有选择地干扰病理细胞运动，如肿瘤细胞的侵袭和增殖，以及细胞膜和细胞核之间的病毒运输的更好的方法。

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.