Effect of hypertrophy mutation on kinetics of single cardiac myosin molecule

描述(由申请者提供)：我的长期目标是建立一个成功的学术生涯，成为一名独立资助的应用单分子荧光的生物物理学家。这里提出的K25奖将为我提供机制，让我从实验物理背景转到这一研究领域。具体地说，我已经提出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)引起的肌球蛋白交叉桥构象的动态变化，以便对抗这些变化。这是我在高级荧光技术方面的博士后工作和我在肌肉功能方面的合作项目的非常直接的延伸。单分子检测对这个项目至关重要，因为肌球蛋白跨桥独立旋转。因此，当从交叉桥的集合测量偏振荧光时，信号从检测体积中所有标记的交叉桥的单独信号与背景贡献的叠加而变得加扰。已经在体外研究了单个肌球蛋白跨桥的动力学，但在分子拥挤一定会产生影响的情况下，体外行为是否会相同还不是很明显。我建议通过以下方式获得肌球蛋白交叉桥的体外单分子检测：(1)将现有的时间分辨共聚焦显微镜转换为受激发射耗竭(STED)超分辨率显微镜，(2)评估荧光纳米钻石(FND)用于单分子的使用，体外肌肉测量，以及最后(3)使用前两个阶段开发的方法来确定FHC诱导的动力学变化。STED技术是将共聚焦显微镜的焦体积限制在一个足够小的区域，使得一次只能观察到一个标记的肌球蛋白分子，并将背景贡献降至最低是必要的。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限制的点扩散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会很麻烦，因为当分子在亮和暗状态之间振荡时，它们容易发光闪烁。更麻烦的是，有机荧光团的光稳定性不是很好--它们在很短的时间内就容易发生光漂白。更高的激光功率加剧了这种效应，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，它不会光眨眼，并且已经被证明几个小时都是光稳定的。它们将通过在这个拟议的项目过程中开发的一种新程序连接到肌球蛋白上。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一肌球蛋白分子的动力学。2011年，我在德克萨斯基督教大学获得凝聚态物理博士学位，并立即过渡到北得克萨斯大学健康科学中心的博士后职位。在这里，我在开发生命科学的先进荧光技术方面发挥了极大的成效。我的出版记录证明了我有能力将我的物理培训转化为与生物相关的工作，以及我对肌肉动力学研究的广泛参与，即使只是到目前为止的技术角色。K25奖将允许必要的生物学课程和指导的实验室工作，以充分发展成为一名敬业的，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.