权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Computational and Single-Molecule Characterization of Kinesin's Power Stroke

驱动蛋白动力冲程的计算和单分子表征

基本信息

批准号：
7357447
负责人：
Wonmuk Hwang
金额：
$ 18.59万
依托单位：
TEXAS ENGINEERING EXPERIMENT STATION
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-04-01 至 2010-03-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Kinesin is a biped motor protein that walks along microtubule tracks in a cell and performs diverse tasks, including intracellular cargo transport and cell division. To date, it is the smallest known processive motor that directly converts the chemical energy of ATP into mechanical energy. A deeper insight into how kinesin functions is thus not only important for advancing fundamental knowledge of molecular motors, but also critical for developing novel therapeutics against diseases involving impaired intracellular transport. Although past biochemical, biophysical, and structural experiments revealed a significant amount of information about kinesin, the basic mechanism by which it operates as a mechanical amplifier to generate a walking stroke remains unknown. To elucidate the mechanism, it will be critical to develop a synergistic approach combining experimental manipulation of individual kinesin molecules and a computational model based on its atomistic structure. Only through such a combined approach will it be possible to find the molecular physical principle that governs the actual walking motion. Our recent molecular dynamics simulation identified the mechanical element responsible for kinesin's power stroke, which we named the cover strand. It works by assisting kinesin's leg, the neck linker, through forming or breaking a bundle with it depending on kinesin's mechanochemical cycle. Formation of the cover-neck bundle results in a forward conformational bias that generates the power stroke. To validate this experimentally, kinesin mutants missing the cover strand will be constructed and tested using single molecule optical trapping force measurements. At the same time, a computational model of the entire kinesin-microtubule complex will be constructed so that kinesin's whole walking step can be investigated in atomistic detail. Response of the mutant kinesin in the single molecule experiments will be interpreted using computational models. In this way, experiments will be used to refine models, while simulation will be used to interpret experimental data and further design new experiments. Such a tight coupling between experimentation and simulation will provide a clear molecular level mechanistic picture of kinesin motility, upon which a host of other motor proteins will be investigated as our long-term goal. Relevance: Deeper understanding of kinesin motility will enable better control of its behavior, which will lead to novel therapeutics that target kinesin-mediated transport. Our combined approach between computational modeling of macromolecular complexes and single-molecule manipulation experiment will also be a platform upon which a range of subcellular motor processes of biomedical importance will be investigated.

描述（由申请人提供）：驱动蛋白是一种在细胞中沿着沿着微管轨道行走并执行多种任务的神经马达蛋白，包括细胞内货物运输和细胞分裂。迄今为止，它是已知的最小的直接将ATP的化学能转化为机械能的加工马达。因此，深入了解驱动蛋白的功能不仅对推进分子马达的基础知识很重要，而且对开发针对涉及细胞内转运受损的疾病的新疗法也至关重要。虽然过去的生物化学，生物物理学和结构实验揭示了大量关于驱动蛋白的信息，但它作为机械放大器产生行走行程的基本机制仍然未知。为了阐明机制，这将是至关重要的，以开发一个协同的方法相结合的实验操作的单个驱动蛋白分子和基于其原子结构的计算模型。只有通过这样一种结合的方法，才有可能找到控制实际行走运动的分子物理原理。我们最近的分子动力学模拟确定了负责驱动蛋白动力行程的机械元件，我们将其命名为覆盖链。它的工作原理是协助驱动蛋白的腿，颈部连接器，通过形成或打破与它取决于驱动蛋白的机械化学循环束。盖颈束的形成导致产生动力冲程的正向构象偏置。为了通过实验验证这一点，将构建缺失覆盖链的驱动蛋白突变体，并使用单分子光学捕获力测量进行测试。与此同时，整个驱动蛋白-微管复合物的计算模型将被构建，以便驱动蛋白的整个行走步骤可以在原子细节研究。将使用计算模型解释单分子实验中突变驱动蛋白的响应。通过这种方式，实验将用于改进模型，而模拟将用于解释实验数据并进一步设计新的实验。实验和模拟之间的这种紧密耦合将提供一个清晰的分子水平的驱动蛋白运动机制的图片，在此基础上，主机的其他马达蛋白将作为我们的长期目标进行研究。相关性：对驱动蛋白运动性的深入了解将能够更好地控制其行为，这将导致靶向驱动蛋白介导的转运的新疗法。我们的大分子复合物的计算建模和单分子操作实验之间的结合方法也将是一个平台，在此基础上，一系列的生物医学重要性的亚细胞运动过程将被调查。