MECHANICS OF KINESIN--MICROTUBULE-BASED MOTOR PROTEIN

拟议研究的长远目标是了解 化学机械转导的物理化学， 高能磷酸键中所含的化学能与机械能 为细胞内运动提供动力的能量。 这一过程完成了 被称为马达蛋白的几种酶，包括来自肌肉的肌球蛋白， 细胞、纤毛和鞭毛的动力蛋白、细胞质动力蛋白和驱动蛋白 从真核细胞中分离出来。 目前的模型，如十字桥 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丝的反应 在肌球蛋白和微管的情况下，动力蛋白和 驱动蛋白 在与纤维结合后，马达蛋白被认为是 经历一个构象变化，动力冲程，产生一个 运动的增量。 然后蛋白质释放细丝， 在沿丝沿着的另一个位点重新结合并开始另一个循环。 所提出的实验的具体目的是直接测试这种 通过对转导反应进行机械测量， 单分子水平。 微管在表面的运动 用纯化的驱动蛋白以低密度包被，将通过暗- 现场显微镜 调查员以前使用的特殊仪器 为了研究耳毛细胞中的力敏离子通道， 用于施加力和测量亚纳米位移 精确到毫秒级 一个驱动蛋白 一个ATP分子水解后，一个ATP分子移动微管， 被确定。 在用单克隆抗体表征微管的运动后， 驱动蛋白分子，几种驱动蛋白之间相互作用的性质 分子移动一个微管由单个驱动蛋白分子，性质 几个驱动蛋白分子之间的相互作用， 微管将被研究，以预测大的行为， 运动蛋白的组装，如在肌肉和纤毛中发现的那些。 由于驱动蛋白和蛋白质在结构和生化上的相似性， 其他马达蛋白，阐明分子事件的基础 驱动蛋白的转导应该会显着增加对 细胞的运动能力 希望这一理解能导致 更合理地治疗肌肉疾病，如心脏病，或 涉及选择性干扰病理性细胞的更好方法， 运动，如肿瘤细胞的侵袭和增殖，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.