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

描述（由申请人提供）：Kinesin是一种两足运动蛋白，在细胞中沿着微管轨道行走并执行多种任务，包括细胞内货物运输和细胞分裂。迄今为止，它是已知的最小的将ATP的化学能直接转化为机械能的过程马达。因此，深入了解运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运输受损的疾病的新疗法也至关重要。尽管过去的生化、生物物理和结构实验揭示了大量关于运动蛋白的信息，但它作为机械放大器产生行走中风的基本机制仍然未知。为了阐明这一机制，关键是要发展一种协同方法，将单个驱动蛋白分子的实验操作与基于其原子结构的计算模型相结合。只有通过这种结合的方法，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发现了驱动蛋白动力冲程的机械元件，我们将其命名为覆盖链。它的工作原理是根据肌动蛋白的机械化学循环，帮助肌动蛋白的腿部，即颈部连接器，与肌动蛋白形成或断开一个束。盖颈束的形成导致正向构象偏差，从而产生动力冲程。为了在实验上验证这一点，将构建缺少覆盖链的激酶突变体，并使用单分子光学捕获力测量进行测试。同时，将构建整个马达蛋白-微管复合物的计算模型，以便对马达蛋白的整个行走步骤进行原子细节的研究。突变激酶在单分子实验中的反应将用计算模型来解释。这样，实验将用于完善模型，而模拟将用于解释实验数据并进一步设计新的实验。这种实验和模拟之间的紧密耦合将提供一个清晰的分子水平运动蛋白的机制图像，在此基础上，许多其他运动蛋白将作为我们的长期目标进行研究。

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.