Gravitational Force Spectroscopy of Single Myosin Molecules

单个肌球蛋白分子的重力光谱

• 批准号: 0842736
• 负责人: Douglas Root
• 金额: $ 23.14万
• 依托单位: University of North Texas
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0842736&HistoricalAwards=false
• 关键词: Gravitational; Force; Spectroscopy; Single; Myosin

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).The coiled coil of myosin is among the longest in nature and contains functionally diverse elements that will be characterized using a newly developed gravitational force spectrometer that facilitates the application of subpiconewton forces to specific regions of single molecules. The subpiconewton forces can be applied at multiple orientations, while the absolute length of a single molecule under force can be measured with a precision in the nanometer range. Dynamic changes in this molecular length can also be quantified at speeds in the millisecond time scale. Using this gravitational force spectrometer, several fundamental hypotheses will be tested about the dynamics of different regions in the coiled coil and theirroles in the functions of myosin molecules. The first hypothesis examines whether the coiled coil stretches more readily due to forces applied perpendicular rather than parallel to the long axis of the coiled coil. If so, the associated stretching could facilitate the properly oriented binding of the myosin head to actin, since in muscle, the myosin coiled coil is perpendicular to the direction of the crossbridge joining thick and thin filaments. The second hypothesis tests whether the hinge regions are more susceptible to stretching under force than other regions of the coiled coil. If the hinges do stretch preferentially in response to the application of subpiconewton forces, then these elastic elements could be an important relay in the transmission of force from the power stroke in the myosin head to filament sliding and the development of tension in muscle which has been implicated from physiological data of muscle contractions. In the third hypothesis, intermolecular interactions of coiled coils between myosin molecules or myosin binding proteins will be carried out to affect the coiled coil's susceptibility to subpiconewton or piconewton forces. These experiments will determine the extent to which the myosin's coiled coil susceptibility to force can be modulated by binding events that are likely to occur in vivo.In addition to some of its unique measuring capabilities, the gravitational force spectrometer is more affordable than other equipment suitable for single molecule assays, so a broader availability for its usage may be possible especially in areas of educational instruction. It is feasible to equip student laboratories with multiple workstations of these devices which would enable hands-on experiences with single molecule assays for science students. To facilitate this possibility, instructional laboratory units will be written with introductory and background descriptions, methods, principles of setup and operation, description of data analysis and acquisition, study questions, key words, and learning outcomes. These instructional laboratoryunits will be evaluated and distributed to encourage the incorporation of single molecule assays into student laboratories which will better prepare future scientists to exploit these developing technologies.

该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。肌球蛋白的盘绕线圈是自然界中最长的线圈之一，包含功能多样的元素，将使用新开发的引力谱仪进行表征，该谱仪有助于将亚皮牛顿力应用于单个分子的特定区域。亚皮牛顿力可以在多个方向上施加，而在力作用下的单个分子的绝对长度可以在纳米范围内精确测量。分子长度的动态变化也可以在毫秒时间尺度上进行量化。利用这台重力谱仪，我们将测试线圈中不同区域的动力学及其在肌球蛋白分子功能中的作用的几个基本假设。第一个假设检查是否卷曲的线圈拉伸更容易由于力施加垂直而不是平行于卷曲线圈的长轴。如果是这样，相关的拉伸可以促进肌凝蛋白头部与肌动蛋白的正确定向结合，因为在肌肉中，肌凝蛋白卷曲的线圈垂直于连接粗丝和细丝的交叉桥的方向。第二个假设测试是否铰链区域更容易受到拉伸下的力比其他区域的卷曲线圈。如果铰链确实在亚皮牛顿力的作用下优先拉伸，那么这些弹性元件可能是一个重要的接力器，从肌凝蛋白头部的力量传递到细丝滑动和肌肉张力的发展，这与肌肉收缩的生理数据有关。在第三种假设中，将进行肌球蛋白分子或肌球蛋白结合蛋白之间的螺旋线圈分子间相互作用，以影响螺旋线圈对亚皮牛顿或皮牛顿力的敏感性。这些实验将确定肌凝蛋白卷曲的线圈对力的敏感性在多大程度上可以通过体内可能发生的结合事件来调节。除了一些独特的测量能力外，重力光谱仪比其他适合单分子分析的设备更便宜，因此它的使用范围更广，特别是在教育教学领域。为学生实验室配备这些设备的多个工作站是可行的，这将使理科生能够实践单分子分析。为了促进这种可能性，教学实验单元将包括介绍和背景描述、方法、设置和操作原则、数据分析和获取描述、研究问题、关键词和学习成果。这些教学实验室单元将被评估和分发，以鼓励将单分子分析纳入学生实验室，这将更好地为未来的科学家利用这些发展中的技术做好准备。