Computational and single molecule analysis of kinesin's atomistic machinery

驱动蛋白原子机制的计算和单分子分析

• 批准号: 7920016
• 负责人: Wonmuk Hwang
• 金额: $ 22.56万
• 依托单位: TEXAS ENGINEERING EXPERIMENT STATION
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7920016
• 关键词: ATP phosphohydrolase; Address; Adenosine Triphosphate; Affect; Affinity; Arts; Be++ element; Behavior; Beryllium; Binding; Binding Sites; C-terminal; Cell division; Characteristics; Computer Simulation; Diffusion; Disease; Docking; Elements; Event; Free Energy; Funding; Generations; Goals; Head; Intracellular Transport; Kinesin; Kinetics; Knowledge; Lead; Macromolecular Complexes; Measures; Mechanics; Mediating; Microtubules; Modeling; Molecular Motors; Motion; Motor; Mutation; N-terminal; Names; Neck; Nucleotides; Outcome; Pathway interactions; Power stroke; Process; Proteins; Regulation; Role; Series; Specificity; Spectrum Analysis; System; Testing; Thermodynamics; Tubulin; Walking; Work; base; cell motility; design; dimer; flexibility; insight; molecular dynamics; mutant; new therapeutic target; novel therapeutics; optical traps; post stroke; public health relevance; research study; simulation; single molecule; success

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Deeper understanding of kinesin motility will enable better control of its behavior and motility characteristics, which will lead to novel therapeutics that target kinesin-mediated transport. Our combined approach of computational modeling of macromolecular complexes and single-molecule manipulation experiment provides a platform upon which a range of subcellular motor processes of biomedical importance will be investigated.

描述（由申请人提供）：驱动蛋白是已知最小的线粒体马达蛋白，其使用ATP作为燃料沿微管轨道沿着行进。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分裂。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。虽然过去的研究揭示了其整体运动特性的许多重要方面，但其步进运动的物理机制仍不清楚。研究驱动蛋白运动性或马达蛋白的主要困难是分子动态地感知并产生运动力，这很难仅基于静态结构图来考虑。为了研究原子细节的动力学方面，我们采取了分子动力学模拟和单分子实验之间的协同方法。利用分子动力学模拟，我们发现驱动蛋白通过折叠一个结构域产生力，我们将其命名为盖颈束。虽然所提出的机制是由我们的单分子运动实验测试驱动蛋白突变体的设计，以产生更少的力的支持，实验导致进一步的问题，关于能量的力的产生，以及在整个驱动蛋白机械化学循环的力产生步骤的作用。此外，我们的初步模拟确定了驱动蛋白运动性的另外两个关键方面：（1）机械应变通过马达头传递以调节核苷酸亲和力的结构途径，这对于马达头协调是重要的，以及（2）微管的C-末端柔性E-钩结构域在偏置马达头的轨迹中的动态作用，这对驱动蛋白如何进行步骤至关重要。这些问题将通过进一步的模拟进行深入研究。将产生突变驱动蛋白，专门改变模拟中发现的物理机制，并使用最先进的光学陷阱系统进行实验测试。这项工作的结果将提供一个更清晰的原子图像的力学基础驱动蛋白运动。以我们之前的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作用来开发，这将是我们研究其他马达蛋白的基础，作为我们的长期目标。公共卫生关系：对驱动蛋白运动性的深入了解将使其行为和运动特性得到更好的控制，这将导致靶向驱动蛋白介导的转运的新疗法。我们将大分子复合物的计算建模和单分子操纵实验结合起来，为研究一系列具有生物医学重要性的亚细胞运动过程提供了一个平台。