Computational and single molecule analysis of kinesin's atomistic machinery

项目摘要 驱动蛋白是已知的最小的运动蛋白，其使用ATP作为燃料沿着沿着 微管径迹它的正常功能对于许多重要任务至关重要，包括细胞内货物运输和细胞内运输。 细胞分裂因此，更深入地了解驱动蛋白的功能不仅对推进基础研究很重要， 分子马达的知识，而且对于开发针对疾病的新疗法也至关重要， 受损的细胞内转运。虽然过去的进展揭示了其全球动力的许多重要方面 特征，其步进运动的物理机制尚不清楚。学习中的一个主要困难 驱动蛋白运动性或一般的马达蛋白，是该分子动态地感知并产生力， 移动，这是很难设想的基础上静态结构图片。为了研究动态方面， 在原子细节上，我们采用了分子动力学模拟和单分子模拟之间协同方法 实验使用分子动力学模拟，我们发现驱动蛋白通过折叠一个 域，我们称之为盖颈束。虽然建议的机制是由我们的单一支持- 分子运动实验测试驱动蛋白突变体，旨在产生更少的力量，实验导致， 关于力产生的能量学以及力产生步骤在 整个驱动蛋白机械化学循环。此外，我们的初步模拟确定了另外两个关键的 驱动蛋白运动性方面：（1）机械应变通过马达传递的结构途径 头调节核苷酸亲和力，这是重要的运动头协调，和（2）动态作用 微管的C-末端柔性E-钩结构域在偏置马达头的轨迹中的作用， 这对驱动蛋白如何迈出一步至关重要。这些问题将通过进一步的模拟进行深入研究。突变体 将产生驱动蛋白，其特异性地改变在模拟和实验中发现的物理机制。 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个更清晰的原子图 驱动蛋白运动的机制。以我们以前的R21资助项目为先导， 工作将通过实验和模拟之间的强大协同作用来发展，这将是基础， 我们的长期目标是研究其他马达蛋白。

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.