MECHANICS OF KINESIN--MICROTUBULE-BASED MOTOR PROTEIN

拟议研究的长期目标是了解 化学-机械转导的物理化学，转化 高能磷键中包含的化学能与机械能 用于为细胞内运动提供动力的能量。这个过程就完成了 通过几种称为马达蛋白的酶，其中包括来自肌肉的肌球蛋白 纤毛和鞭毛中的动力蛋白，以及细胞质动力蛋白和动力蛋白 从真核细胞中分离出来。目前的型号，如交叉桥 模型，假设潜在的转导过程是一个循环 马达分子与细胞骨架聚合物--肌动蛋白细丝的反应 在肌球蛋白和微管的情况下，动力蛋白和 激动素。在与细丝结合后，马达蛋白被认为是 经历一种构象变化，即产生一种 运动的增量。然后，蛋白质在此之前释放细丝 在沿着细丝的另一个位置重新结合，并启动另一个循环。 提议的实验的具体目的是直接测试这样的 通过对转导反应进行力学测量来建立模型 单分子水平。微管在表面上的运动 在低密度下涂上纯化的激动素将在黑暗中显现- 现场显微镜。调查员以前使用的特殊仪器 为了研究耳朵毛细胞中的力敏感离子通道，将 用于施力和测量亚纳米级的位移 毫秒时间刻度的精度。一次运动的距离 分子在三磷酸腺苷分子的水解液中移动微管 要下定决心。在对微管的运动进行单项表征后 动蛋白分子，几种动蛋白相互作用的本质 分子通过单个运动蛋白分子移动一个微管，自然界 几个运动蛋白分子之间的相互作用 将对微管进行研究，以预测大鼠的行为 马达蛋白质的集合，如在肌肉和纤毛中发现的那些。 因为激动素和肌动蛋白在结构和生化上的相似性 其他马达蛋白，阐明潜在的分子事件 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。希望这种理解可能会导致 到更合理的肌肉疾病治疗，如心脏病，或 更好的选择性干扰病理细胞的方法 肿瘤细胞的侵袭和增殖等运动，以及 病毒在细胞膜和细胞核之间的运输。

The long-term objective of the proposed studies is to understand the physical chemistry of chemomechanical transduction, the conversion of chemical energy contained in high-energy phosphate bonds to mechanical energy used to power intracellular movement. This process is accomplished by several enzymes, termed motor proteins, that include myosin from muscle cells, dynein from cilia and flagella, and cytoplasmic dynein and kinesin from eukaryotic cells in general. Current models, such as the crossbridge model, postulate that the underlying transduction process is a cyclic reaction of a motor molecule with a cytoskeletal polymer, an actin filament in the case of myosin and a microtubule in the instances of dynein and kinesin. After binding to the filament, the motor protein is thought to undergo a conformational change, the power stroke, that produces an increment of movement. The protein then releases the filament before rebinding at another site along the filament and initiating another cycle. The specific aim of the proposed experiments is to test directly such models by making mechanical measurements of the transduction reaction at the single-molecular level. The movement of microtubules across surfaces coated at low density with purified kinesin will be visualized by dark- field microscopy. Special apparatus, previously used by the investigator to study force-sensitive ion channels in hair cells of the ear, will be used to exert forces and to measure displacements with subnanometer precision on a millisecond timescale. The distance that a single kinesin molecule moves a microtubule upon the hydrolysis of a molecule of ATP will be determined. After characterizing the movement of microtubules by single kinesin molecules, the nature of the interactions between several kinesin molecules moving one microtubules by single kinesin molecules, the nature of the interactions between several kinesin molecules moving one microtubule will be studied in order to predict the behavior of large assemblies of motor proteins such as those found in muscles and cilia. Because of the structural and biochemical similarities between kinesin and other motor proteins, the elucidation of the molecular events underlying 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.