Kinetic mechanisms of amino acid transporters

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作用机制 指细胞内或细胞外的物质，如关键营养物质氨基酸和葡萄糖。这个 对这些蛋白质的机制的了解反过来使我们能够追寻致病 研究某些疾病过程的机制，并开发治疗方法。一般来说，运输的基础是什么？ 过程是转运蛋白的一系列构象变化。这项提案的目标是适用于 我们新开发的基于高分辨率荧光偏振显微镜的方法来确定 发现一种具有重要生物医学意义的转运蛋白构象变化的能量学和动力学 在一些致病细菌中。 结构生物学产生了丰富的蛋白质结构，揭示了蛋白质的结构基础 功能。然而，对蛋白质分子的充分理解必须包括它的空间和时间。 特点。因此，我们需要超越在近原子尺度上研究蛋白质在 以静态的方式，并以动态的方式研究它。然而，所需的实验信息 由于缺乏相对通用的可靠跟踪方法，因此常常缺乏关于蛋白质动力学的信息 蛋白质在埃尺度上的快速构象变化。通常，这样的微小更改可以可靠地 只能用结晶学或低温电子显微镜等结构技术进行定量解析， 不幸的是，缺乏时间分辨率。另一方面，传统的光学显微镜可能是时间分辨的，但 它的空间分辨率仍然太低，无法分辨埃尺度的蛋白质构象变化。 最近，我们成功地解决了毫秒和埃的蛋白质构象变化 通过检查粘贴在检查的选定片段上的单个荧光标签的各向异性来进行缩放 蛋白质，已知在蛋白质的每种晶体结构状态中采用唯一的取向。有一个州- 最先进的偏光显微镜和分析，我们已经获得了有效的角度分辨率为5- 10°。在这个范围内，一个平均大小的蛋白质分子的旋转运动将导致1.7-3.5？ 和弦距离的变化。将这种方法应用于转运蛋白，我们将确定能量学。 以及作为其运输功能基础的构象变化的动力学。整合由此产生的动态 带有结构信息的信息最终将产生一个完整的、完整的四维机械 解释转运蛋白行为的模型，其精确度和精确度 潜在的衡量标准。我们提议的研究的成功将改变我们研究动态的方式 包括转运蛋白在内的膜蛋白的机制，并加速从电流， 从静态的结构生物学到动态的结构生物学。

Project Summary Our long-term goal is to understand the mechanism of a class of protein molecules in the membranes of cells, which transport substances in or out of cells, such as the key nutrients amino acids and glucose. The knowledge regarding the mechanisms of these proteins in turn enable us to pursue the pathogenic mechanisms of certain disease processes and to develop therapies. Generally, what underlies the transporting process is a series of conformational changes of the transporter protein. The goal of this proposal is to apply our newly developed high-resolution fluorescence-polarization-microscope-based method to determine the energetics and dynamics of the conformational changes of a biomedically important transporter protein found in some pathogenic bacteria. Structural biology has yielded abundant protein structures, revealing the structural basis of protein functions. However, a full understanding of a protein molecule must include both its spatial and temporal characteristics. We thus need to go beyond studying the behaviors of a protein on a near-atomic scale in a static manner, and study it in a dynamic manner instead. However, the required 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. Generally, such small changes can be reliably and quantitatively resolved only with such structural techniques as crystallography or Cryo-EM, which, unfortunately, lack time resolution. Conventional light microscopy, on the other hand, may be time-resolved but its spatial resolution had remained too low to resolve angstrom-scale protein conformational changes. Recently, we have successfully resolved protein conformational changes on millisecond-and-angstrom scales by examining anisotropy of a single fluorescent label attached to a chosen segment in an examined protein, which is known to adopt a unique orientation in each crystal structural state of the protein. With a state- of-the-art polarization microscope and analytic analyses, 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. Applying this method to the transporter protein, we will determine the energetics and kinetics of conformational changes that underlie its transporting function. Integrating the resulting dynamic information with structural information will ultimately yield an integrated, full four-dimensional mechanistic model that accounts for the behaviors of the transporter protein, at the precision and accuracy of the underlying measurements. Success of our proposed study will transform the way we investigate the dynamic mechanisms of membrane proteins including transporters, and accelerate the transition from the current, mostly static approach of structural biology to dynamic structural biology.