Angstrom-scale structural dynamics of a potassium channel

项目摘要 我们的长期目标是了解一类蛋白质分子在细胞膜中的机制， 这使得离子能够流入或流出细胞，从而产生重要的细胞电信号。《知识》 关于这些蛋白质的机制，称为离子通道，形成了我们 探索某些疾病过程的致病机制，并开发治疗方法。一般而言， 通道的功能由特定的信令过程来调节，这些调节的基础是什么 蛋白质构象变化。这项提议的目标是进一步发展荧光偏振 一种在三维(3D)中精确跟踪附着在蛋白质上的荧光团的显微镜方法 埃和毫秒尺度，并将该方法应用于构象动力学的研究 细菌Trk通道，这是对抗某些耐药肠道细菌的新靶点。 结构生物学产生了丰富的蛋白质结构。然而，对蛋白质的充分理解 分子必须同时包括其空间和时间特征。因此，我们需要超越对蛋白质的研究 原子尺度上的静态结构，并研究其在埃和毫秒尺度上的动力学。到目前为止， 关于蛋白质动力学的实验信息往往是缺乏的，这是因为缺乏相对通用的 可靠跟踪蛋白质的埃尺度构象快速变化的方法。通常情况下，这样的小 只有使用结晶学或其他结构技术才能可靠和定量地解决变化问题 冷冻-EM，不幸的是，它缺乏时间分辨率。光学显微镜可能是时间分辨的，但它的空间 分辨率普遍太低，无法分辨埃尺度的蛋白质构象变化。 最近，我们成功地解决了发生在毫秒和- Angstrom通过检测附着在被检测蛋白质上的荧光团的发射极化来衡量。 有了最先进的荧光偏振显微镜和我们组装的分析包，我们 获得了5-10°的有效角度分辨率。在这个范围内，蛋白质分子的旋转运动 平均大小的变化会导致弦距变化1.7-3.5？在这里，我们将继续发展 这种方法，并证明了它的适用性超越了用于开发该方法的分子，此外 获得生物医学科学方面的重要知识。用这种方法，我们将确定能量学和 Trk通道功能背后的蛋白质构象变化的动力学。集成由此产生的 具有可用结构信息的动态信息将产生一个机械性的时空4D模型 解释了通道在毫秒和埃尺度上的行为。我们研究的成功将改变 我们研究蛋白质的动力学机制，并加速从 目前，实验结构生物学的方法大多是静态的，而现在的方法是综合的动态方法。

Project Summary Our long-term goal is to understand the mechanism of a class of protein molecules in cell membranes, which enable ions to flow in or out of cells and thereby generate vital cellular electric signals. The knowledge regarding the mechanisms of these proteins, called ion channels, form the essential scientific basis for us to pursue the pathogenic mechanisms of certain disease processes and to develop therapies. Generally, the functions of channels are regulated by specific signaling processes, and what underlie these regulations are protein-conformational changes. The goal of this proposal is to further develop a fluorescence-polarization microscopy method to accurately track in three-dimensions (3D) a fluorophore attached to a protein on an angstrom-and-millisecond scale, and to apply this method to the investigation of conformational dynamics of the bacterial Trk channel, which is a new target to combat certain antibiotics-resistant enterobacteria. Structural biology has yielded abundant protein structures. However, a full understanding of a protein molecule must include both its spatial and temporal features. We thus need to go beyond studying a protein's static structures on an atomic scale and study its dynamics on angstrom-and-millisecond scales. Thus far, the experimental information about protein dynamics is often lacking, due to the absence of relatively general methods for reliably tracking rapid angstrom-scale conformational changes of a protein. Typically, such small changes can be reliably and quantitatively resolved only with structural techniques such as crystallography or Cryo-EM, which, unfortunately, lack time resolution. Light microscopy may be time-resolved but its spatial resolution had remained generally too low to resolve angstrom-scale protein conformational changes. Recently, we have successfully resolved protein conformational changes occurring on millisecond-and- angstrom scales by examining emission polarization of a fluorophore attached to a protein under examination. With a state-of-the-art fluorescence-polarization microscope and an analytic package that we put together, we have achieved an effective angle resolution of 5-10°. Over this range, a rotational motion of a protein molecule of an average size would cause a 1.7 - 3.5 Å change in the chord distance. Here, we will continue to develop this method and demonstrate its applicability beyond the molecule used to develop the method, besides acquiring important knowledge in biomedical science. With this method, we will determine the energetics and kinetics of the protein-conformational changes underling the Trk channel's function. Integrating the resulting dynamic information with available structural information will yield a mechanistic spatiotemporal 4D model that accounts for the channel's behaviors on millisecond-and-angstrom scales. Success of our study will transform the way that we investigate the dynamic mechanisms of proteins, and accelerate the transition from the current, mostly static approach of experimental structural biology to an integrative dynamic approach.