Kinetic mechanisms of amino acid transporters

氨基酸转运蛋白的动力学机制

• 批准号: 10027946
• 负责人: ZHE LU
• 金额: $ 40.5万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10027946
• 关键词: Achievement; Adopted; Agmatine; Amino Acid Transporter; Amino Acids; Anisotropy; Arginine; Bacteria; Behavior; Biological Transport; Carrier Proteins; Cell membrane; Cells; Cellular Membrane; Characteristics; Complement; Cryoelectron Microscopy; Crystallization; Crystallography; Data; Development; Disease; Elements; Equilibrium; Event; Fluorescence Polarization; Foundations; Four-dimensional; Future; Glucose; Glucose Transporter; Goals; Human; Individual; Intestines; Investigation; Kinetics; Knowledge; Label; Lead; Light Microscope; Measurement; Measures; Membrane; Membrane Proteins; Methods; Microscope; Microscopy; Modeling; Molecular Conformation; Monitor; Motion; Movement; Nutrient; Pathogenicity; Probability; Process; Protein Conformation; Protein Dynamics; Proteins; Protons; Radial; Resolution; Rotation; Science; Series; Site; Space Perception; Specific qualifier value; Stomach; Structural Biologist; Structural Models; Structure; System; Techniques; Thinking; Time; Transport Process; alpha helix; base; behavioral study; conformational conversion; design; fluorophore; kinetic model; light microscopy; millisecond; molecular dynamics; pathogenic bacteria; protein function; protein structure; spatiotemporal; structural biology; success; temporal measurement; therapy development; three dimensional structure; virtual

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.

项目摘要 我们的长期目标是了解膜中一类蛋白质分子的机制 它是细胞的一个重要组成部分，负责将物质运入或运出细胞，如关键的营养素氨基酸和葡萄糖。的 关于这些蛋白质的机制的知识反过来使我们能够追求致病性， 某些疾病过程的机制和开发治疗方法。一般来说，运输的基础是什么？ 这一过程是转运蛋白的一系列构象变化。该提案的目的是适用于 我们新开发的高分辨率荧光偏振显微镜为基础的方法，以确定 能量学和动力学的构象变化的生物医学上重要的转运蛋白发现 在某些致病细菌中。 结构生物学已经产生了丰富的蛋白质结构，揭示了蛋白质的结构基础 功能协调发展的然而，对蛋白质分子的全面理解必须包括其空间和时间 特色因此，我们需要超越在近原子尺度上研究蛋白质的行为， 静态地研究，动态地研究。然而，所需的实验信息 由于缺乏相对通用的可靠跟踪方法，通常缺乏有关蛋白质动力学的信息 蛋白质的快速的埃尺度构象变化。一般来说，这种小的变化可以可靠地， 定量解析只能用这样的结构技术，如晶体学或冷冻电镜， 不幸的是，缺乏时间解决方案。另一方面，传统的光学显微镜可以是时间分辨的， 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，我们已经成功地解决了蛋白质构象变化的毫秒和埃 通过检查附着在所检查的细胞中的选定片段上的单个荧光标记的各向异性来缩放。 蛋白质，已知其在蛋白质的每个晶体结构状态中采用独特的取向。一个国家- 最先进的偏光显微镜和分析分析，我们已经实现了5- 10度。在这个范围内，一个平均大小的蛋白质分子的旋转运动将引起1.7 - 3.5 π 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 具有结构信息的信息最终将产生一个完整的四维机械 模型，占转运蛋白的行为，在精度和准确性的 基本的测量。我们提出的研究的成功将改变我们调查动态的方式。 包括转运蛋白在内的膜蛋白的机制，并加速从电流， 从静态结构生物学到动态结构生物学。