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: 8134974
• 负责人: Wonmuk Hwang
• 金额: $ 22.3万
• 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/8134974
• 关键词: ATP phosphohydrolase; Address; Adenosine Triphosphate; Affect; Affinity; Behavior; Beryllium; Binding; Binding Sites; C-terminal; Cell division; Characteristics; Computer Simulation; Diffusion; Disease; Docking; Elements; Event; Free Energy; Funding; Generations; Goals; Head; Health; 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; 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(申请人提供)：Kinesin是已知的最小的两足动物马达蛋白，它使用ATP作为燃料沿着微管轨道连续行走。它的正确功能对许多重要任务至关重要，包括细胞内的货物运输和细胞分裂。因此，更深入地了解Kinesin的功能不仅对于推进分子马达的基础知识非常重要，而且对于开发针对涉及细胞内转运受损的疾病的新疗法也是至关重要的。虽然以往的研究揭示了其全球运动特性的许多重要方面，但其步进运动背后的物理机制仍不清楚。研究运动素运动或马达蛋白质的一个主要困难是分子动态地感知并产生移动的力，这是很难仅基于静态结构图像来考虑的。为了在原子细节上研究动力学方面，我们采取了分子动力学模拟和单分子实验相结合的方法。利用分子动力学模拟，我们发现动蛋白是通过折叠一个结构域来产生力的，我们称之为套颈束。虽然所提出的机制得到了我们的单分子运动学实验的支持，该实验测试了旨在产生较少力的突变体中的运动，但这些实验引发了关于力生成的能量学以及力生成步骤在机械力化学循环中的整个运动步骤中的作用的进一步问题。此外，我们的初步模拟发现了Kinesin运动性的另外两个关键方面：(1)机械应变通过运动头传递以调节核苷酸亲和力的结构路径，这对运动头的协调非常重要；(2)微管的C-末端柔性E-Hook结构域在偏置运动头的轨迹中的动态作用，这对Kinesin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。突变的动蛋白将被产生，专门改变在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。这项工作的结果将为运动学背后的机制提供一个更清晰的原子学图景。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作用来发展，这将是我们作为长期目标研究许多其他马达蛋白质的基础。公共卫生相关性：更深入地了解运动蛋白的运动将使其能够更好地控制其行为和运动特性，这将导致针对运动蛋白介导的运输的新疗法。我们的大分子复合体计算建模和单分子操纵实验相结合的方法提供了一个平台，在此平台上将研究一系列具有生物医学意义的亚细胞运动过程。