Effect of hypertrophy mutation on kinetics of single cardiac myosin molecule

描述（由申请人提供）：我的长期目标是建立一个成功的学术生涯，作为一个独立资助的生物物理学家应用单分子荧光。这里提出的K25奖将为我从实验物理的背景转入这一研究方向提供机制。具体来说，我建议通过偏振荧光的单分子检测来确定家族性肥厚性心肌病（FHC）导致的肌球蛋白交叉桥构象的动态变化，以便可以对抗这些变化。这是我博士后研究高级荧光技术和肌肉功能合作项目的直接延伸。单分子检测对这个项目至关重要，因为肌凝蛋白交叉桥是独立旋转的。因此，当从交叉桥的集合中测量偏振荧光时，信号从检测体积中所有标记交叉桥的单个信号的叠加以及背景贡献中变得混乱。在体外研究了单个肌球蛋白过桥的动力学，但在分子拥挤肯定会产生影响的情况下，这种行为在体外是否相同并不明显。我建议通过以下方式获得肌球蛋白交叉桥的离体单分子检测：(1)将现有的时间分辨共聚焦显微镜转换为受激发射损耗（STED）超分辨显微镜，(2)评估荧光纳米金刚石（FNDs）在肌肉中进行单分子离体测量的使用，最后(3)使用前两个阶段开发的方法来识别FHC引起的动力学变化。STED技术必须将共聚焦显微镜的聚焦体积限制在一个足够小的区域内，以便在任何时候只观察到一个标记的肌球蛋白分子，并且使背景贡献最小化。在目前正在开发或正在使用的许多超分辨率技术中，STED是被选择的技术，因为它是唯一一种可以在避免小于传统显微镜系统的衍射极限点扩展函数的范围内研究快速动力学的技术。然而，有机荧光团的单分子观察可能是麻烦的，因为分子在明暗状态之间振荡时容易发生光闪烁。更麻烦的是，有机荧光团的光稳定性不太好，很容易在短时间内发生光漂白。这种效应被更高的激光功率加剧，这是从单个分子获得足够高的信号所必需的。因此，我们建议使用fnd，它不会光瞬变，并且已被证明在几个小时内都是光稳定的。它们将通过本项目中开发的新程序附着在肌凝蛋白上。随着显微镜和标记问题的充分解决，该项目的第三阶段将研究转基因小鼠健康和患病心脏组织中单个肌球蛋白分子的动力学。我于2011年获得德克萨斯基督教大学凝聚态物理学博士学位，并立即转到北德克萨斯大学健康科学中心担任博士后。在这里，我在为生命科学开发先进的荧光技术方面非常有成效。我的出版记录证明了我有能力将我的物理训练转化为生物学相关的工作，并且我广泛参与了肌肉动力学的研究，如果只是到目前为止的技术角色。K25奖将允许必要的生物课程，并指导实验室工作，以充分发展成为一个专门的，独立的生物物理学家。

DESCRIPTION (provided by applicant): My long term goal is to establish a successful academic career as an independently funded biophysicist applying single-molecule fluorescence. The K25 award proposed here will provide the mechanism for me to transfer into this line of study from my background of experimental physics. Specifically, I have proposed to determine the dynamic changes in conformation of a myosin cross-bridges as a result of Familial Hypertrophic Cardiomyopathy (FHC) via single molecule detection of polarized fluorescence so that these changes may be combatted. This is a very direct extension of my postdoctoral work in advanced fluorescence techniques and my collaborative projects in muscle function. Single molecule detection is of critical importance to this project, because the myosin cross-bridges rotate independently. Thus when polarized fluorescence is measured from an ensemble of cross-bridges, the signal becomes scrambled from the superposition of the individual signals of all labeled cross-bridges in the detection volume along with background contributions. The kinetics of a single myosin cross-bridge have been studied in vitro, but it is not at all obvious that the behavior would be the same ex vivo, when molecular crowding will be sure to have an effect. I propose to obtain ex vivo, single molecule detection of a myosin crossbridge in the following manner by (1) converting an existing, time-resolved, confocal microscope into a Stimulated Emission Depletion (STED), super-resolution microscope, (2) evaluating the use of Fluorescent Nanodiamonds (FNDs) for single molecule, ex vivo measurements in muscle, and finally (3) using the methods developed in the previous two stages to identify changes in kinetics induced by FHC. The STED technique is necessary to confine the focal volume of the confocal microscope to an area small enough that only one labeled myosin molecule is observed at any one time and the background contribution is minimized. Of the many super- resolution techniques currently under development or being employed, STED is the chosen technique, because it is the only one that can allow the study of fast dynamics on a scale that avoids smaller than the diffraction-limited point spread function of conventional microscope systems. However, single molecule observation of an organic fluorophore can be troublesome, as the molecules are prone to photoblinking as they oscillate between light and dark state. Even more troublesome, organic fluorophores are not very photostable-they are prone to photobleaching after only short periods of time. This effect is exacerbated by higher laser powers, which are necessary to obtain a sufficiently high signal from a single molecule. Thus we suggest the use of FNDs, which do not photoblink and have been shown to be photostable for hours. They will be attached to myosin by a new procedure developed in the course of this proposed project. With the microscopy and labeling concerns fully addressed, the third phase of this project will study the kinetics of a single myosin molecul in healthy and diseased heart tissue from transgenic mice. I received a Ph.D. in Condensed Matter Physics from Texas Christian University in 2011, and immediately transitioned to Postdoctoral appointment at the University of North Texas Health Science Center. Here I have been extremely productive in the development of advanced fluorescence techniques for the life sciences. My publishing record demonstrates my ability to translate my physics training into biologically relevant work and my extensively involvement in the investigation of muscle dynamics, if only in a technical role thus far. The K25 award will allow for necessary biological coursework, and mentored lab work to fully develop into a dedicated, independent biophysicist.