MECHANICS OF KINESIN-- A MICROTUBULE-BASED MOTOR PROTEIN

描述：（申请人摘要的逐字记录。）长期目标 一个重要的研究方向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， 驱动蛋白一般来自真核细胞， 从ATP的γ-磷酸键水解成机械功， 为细胞内运输提供动力。 该策略的建议，其中重点是微管为基础的电机 驱动蛋白，是联合收割机高灵敏度的单分子技术， 生物化学和蛋白质工程技术，以确定移动 电动机的部件-弹簧，杠杆和车轴-并了解它们的 协调运动与ATP的水解有关。驱动蛋白是一种进行性的 能够沿着微管沿着多个步骤而不解离的马达。我们 将测试持续合成能力是否是由于机械协调， 通过测量力如何影响驱动蛋白的两个运动域的解离， 从微管中分离出来。假定的弹性元件将 本地化，和一个关键的预测，过桥周期模型将是 通过比较单电机力与弹性力的乘积来测试 元件的刚度和动力行程距离。基于近似 二聚体驱动蛋白的双重对称性，当它的两个头部在同一个 核苷酸状态，我们假设，电力中风是与一个 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。来确定有多紧 化学和机械步骤之间的耦合，我们将测量效果 对ATP水解率的影响。这些结果将被纳入一个 动力学模型，该模型将电机的速度和持续合成能力与 它所携带的载荷和ATP浓度。 由于驱动蛋白，肌球蛋白， 和动力蛋白，阐明了能量背后的分子事件 驱动蛋白的转导应该会显着增加对 细胞的运动能力希望这一谅解能导致 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 细胞膜和细胞核之间的病毒。

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.