Gravitational Force Spectroscopy of Single Myosin Molecules

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。肌球蛋白的卷曲螺旋是自然界中最长的，包含功能多样的元素，其特征在于使用新开发的引力光谱仪，便于将亚微微牛顿力应用于单分子的特定区域。亚微微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以纳米范围内的精度测量。这种分子长度的动态变化也可以以毫秒时间尺度的速度量化。利用这台引力谱仪，我们将检验关于卷曲螺旋中不同区域的动力学以及它们在肌球蛋白分子功能中的作用的几个基本假设。第一个假设检查了卷曲螺旋是否由于垂直于而不是平行于卷曲螺旋的长轴施加的力而更容易拉伸。如果是这样的话，相关的拉伸可以促进肌球蛋白头部与肌动蛋白的正确定向结合，因为在肌肉中，肌球蛋白卷曲的螺旋垂直于连接粗丝和细丝的横桥的方向。第二个假设测试铰链区是否比卷曲螺旋的其他区域更容易在力的作用下拉伸。如果铰链确实优先响应于亚微微牛顿力的施加而伸展，则这些弹性元件可能是从肌球蛋白头部中的动力冲程到细丝滑动的力的传递和肌肉中的张力的发展中的重要中继，这已经从肌肉收缩的生理数据中暗示出来。在第三种假设中，肌球蛋白分子或肌球蛋白结合蛋白之间的卷曲螺旋的分子间相互作用将影响卷曲螺旋对亚微微牛顿或微微牛顿力的敏感性。这些实验将确定肌球蛋白的卷曲螺旋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，重力谱仪比其他适合单分子测定的设备更便宜，因此其更广泛的使用可能是可能的，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，这将使理科学生能够亲身体验单分子测定。为了促进这种可能性，教学实验室单元将编写介绍和背景描述，方法，设置和操作的原则，数据分析和采集的描述，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配，以鼓励将单分子测定纳入学生实验室，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。

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.