Mechanics of Kinesin: a Microtubule-Based Motor Protein

驱动蛋白的力学：一种基于微管的运动蛋白

基本信息

批准号：
6874904
负责人：
Jonathon Howard
金额：
$ 18.36万
依托单位：
INSTITUTE FOR MOLECULAR CELL BIOLOGY
依托单位国家：
美国
项目类别：
财政年份：
1990
资助国家：
美国
起止时间：
1990-06-30 至 2007-03-31
项目状态：
已结题

项目摘要

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.

拟议研究的长期目标是了解运动蛋白的工作原理。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，它们将 ATP 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。该提案的策略重点是基于微管的运动驱动蛋白，将高灵敏度单分子技术与生化和蛋白质工程技术相结合，以期将高灵敏度单分子技术与生化和蛋白质工程技术结合起来识别运动部件（弹簧、水平仪和轴）并了解它们的协调运动如何与 ATP 的水解耦合。驱动蛋白是一种处理马达，能够沿着微管进行许多步骤而不解离。我们将通过测量力如何影响各个头与微管的分离来测试先行性是否是由于驱动蛋白的两个运动域之间的机械协调所致。假定的弹性元件将被本地化，并且将通过将单电机力与弹性元件的刚度和动力冲程距离的乘积进行比较来测试横桥循环模型的关键预测。我们将通过测量不同核苷酸条件下驱动蛋白的扭转刚度来直接确定结合核苷酸的变化是否会改变驱动蛋白两个头部的活动性。基于当其两个头处于相同核苷酸条件时二聚驱动蛋白的大约双重对称性。基于当两个头处于相同核苷酸状态时二聚驱动蛋白的近似双重对称性，我们假设动力冲程与一个头相对于另一个头的旋转相关：我们将使用单分子荧光显微镜可视化这种旋转。为了确定化学步骤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。将开发一个动力学模型来将这些机械结果与驱动蛋白的生化综合起来。由于驱动蛋白、肌球蛋白和动力蛋白之间的结构和生化相似性，阐明驱动蛋白能量转导的分子事件应该会显着增加对细胞运动的总体理解。希望这种理解可以带来对心脏病等肌肉疾病的更合理的治疗，或者更好的方法来选择性地干扰病理性细胞运动，例如肿瘤细胞的侵袭和增殖，以及病毒在细胞膜之间的运输和细胞核。