Computational and single molecule analysis of kinesin's atomistic machinery

项目摘要 Kinesin是已知的最小的两足动物马达蛋白，它以ATP为燃料沿着 微管轨迹。它的正确功能对许多重要任务至关重要，包括细胞内货物运输和 细胞分裂。因此，更深入地了解Kinesin是如何发挥作用的不仅对推动 分子马达知识，但也是开发针对以下疾病的新疗法的关键 细胞内转运受损。虽然过去的进展揭示了其全球能动性的许多重要方面 但其步态运动的物理机制尚不清楚。学习中的一个主要困难 运动素运动性或马达蛋白一般是指分子动态地感知并产生力以 移动，这是很难仅仅基于静态的结构图片来思考的。调查动态方面 在原子细节上，我们采取了分子动力学模拟和单分子模拟相结合的方法 做实验。使用分子动力学模拟，我们发现动蛋白通过折叠一个 域，我们将其命名为套颈束。虽然建议的机制由我们的单一机构支持- 分子运动学实验测试旨在产生较少作用力的运动蛋白突变体，这些实验导致 关于力量产生的能量学以及力量产生步骤在#年的作用的进一步问题 机械力化学循环中的整体运动。此外，我们的初步模拟确定了另外两个关键因素 动蛋白运动的几个方面：(1)机械应变通过马达传递的结构路径 头部调节核苷酸亲和力，这对运动头部的协调很重要，以及(2)动态作用 微管的C末端柔性E钩结构域在偏置运动头的轨迹中的作用，这是 对激动素如何迈出一步至关重要。这些问题将通过进一步的模拟得到彻底的研究。突变型 运动蛋白将产生专门改变在模拟和实验中发现的物理机制的运动蛋白 使用最先进的光学陷阱系统进行测试。这项工作的结果将提供一幅更清晰的原子图 作为运动学基础的机械原理。以我们之前由R21资助的项目为先导，拟议的 工作将通过实验和模拟之间的强大协同来发展，这将是 作为我们的长期目标，我们将研究哪些其他马达蛋白。

Project Summary Kinesin is the smallest known biped motor protein that uses ATP as a fuel to walk processively along the microtubule track. Its proper function is critical for many vital tasks including intracellular cargo transport and cell division. 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. While past advances revealed many important aspects on its global motility characteristics, physical mechanism underling its stepping motion remains unclear. A major difficulty in studying kinesin motility or motor proteins in general, is that the molecule dynamically senses and generates force to move, which is difficult to contemplate based on static structural picture only. To investigate the dynamic aspect in atomistic detail, we take a synergistic approach between molecular dynamics simulation and single-molecule experiment. Using molecular dynamics simulations, we discovered that kinesin generates force by folding of a domain, which we named the cover-neck bundle. While the proposed mechanism is supported by our single- molecule motility experiments testing kinesin mutants designed to generate less force, the experiments led to further questions regarding energetics of the force generation as well as the role of the force-generating step in the overall kinesin mechanochemical cycle. Furthermore, our preliminary simulations identified two other crucial aspects of kinesin motility: (1) the structural pathway by which mechanical strain is transmitted through the motor head to modulate the nucleotide affinity, which is important for motor head coordination, and (2) the dynamic role of the C-terminal flexible E-hook domains of the microtubule in biasing the trajectory of a motor head, which is critical for how kinesin makes a step. These issues will be thoroughly investigated by further simulations. Mutant kinesins will be generated that specifically alter the physical mechanism found in simulations, and experimentally tested using state-of-the-art optical trap systems. Outcome of this work will provide a clearer atomistic picture of the mechanics underling kinesin motility. With our previous R21-funded project as a precursor, the proposed work will be developed via strong synergy between experiments and simulations, which will be the basis upon which a host of other motor proteins will be investigated as our long-term goal.