Effect of hypertrophy mutation on kinetics of single cardiac myosin molecule

肥大突变对单个心肌肌球蛋白分子动力学的影响

基本信息

项目摘要

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.
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药理学家应用单分子荧光。这里提出的K25奖将为我提供一种机制,使我从实验物理学的背景转入这一研究领域。具体来说,我已经提出,以确定动态变化的构象的肌球蛋白跨桥作为一个结果,家族性肥厚型心肌病(FHC)通过单分子检测偏振荧光,使这些变化可能会被打击。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一个非常直接的延伸。单分子检测对于该项目至关重要,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,信号由于检测体积中所有标记的交叉桥的单独信号的叠加以及背景贡献沿着而变得杂乱。已经在体外研究了单个肌球蛋白跨桥的动力学,但是当分子拥挤肯定会产生影响时,离体行为是否相同根本不明显。我建议以以下方式获得肌球蛋白横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耗尽(STED)超分辨率显微镜,(2)评估荧光纳米金刚石(FND)用于肌肉中单分子离体测量的用途,最后(3)利用前两个阶段发展的方法确定FHC诱导的动力学变化。STED技术必须将共聚焦显微镜的聚焦体积限制在足够小的区域内,使得在任何一个时间仅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是所选择的技术,因为它是唯一一种可以允许在避免小于传统显微镜系统的衍射限制点扩散函数的尺度上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,因为分子在亮和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是很好,它们很容易在短时间内发生光漂白。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,它不闪烁,并已被证明是耐光数小时。它们将通过在本拟议项目过程中开发的新程序附着到肌球蛋白上。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个肌球蛋白分子的动力学。我获得了博士学位。2011年,他在德克萨斯基督教大学获得了凝聚态物理学博士学位,并立即在北德克萨斯大学健康科学中心获得博士后任命。在这里,我在生命科学的先进荧光技术的发展方面非常富有成效。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,以及我广泛参与肌肉动力学的研究,即使到目前为止只是在技术方面。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,独立的生物制药师。

项目成果

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Ryan Michael Rich其他文献

Ryan Michael Rich的其他文献

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{{ truncateString('Ryan Michael Rich', 18)}}的其他基金

Effect of hypertrophy mutation on kinetics of single cardiac myosin molecule
肥大突变对单个心肌肌球蛋白分子动力学的影响
  • 批准号:
    8768006
  • 财政年份:
    2014
  • 资助金额:
    $ 9.38万
  • 项目类别:

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