Linking proton pumping to hydride transfer: mechanism of transhydrogenase

将质子泵浦与氢化物转移联系起来：转氢酶的机制

• 批准号: 1616874
• 负责人: Robert Gennis
• 金额: $ 82.75万
• 依托单位: University of Illinois at Urbana-Champaign
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-15 至 2020-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1616874&HistoricalAwards=false
• 关键词: Linking; proton; pumping; hydride; transfer

One essential feature of all life is the ability to extract energy from the environment and convert it to useful work. Humans, other animals, plants and microbes all share a set of strategies for conserving and converting energy from one form to another. A central element in these strategies is to generate and then utilize an electrical voltage across a membrane. Energy in one form is used to separate positive and negative charges across a membrane, and then this voltage is used to drive a current of ions that is used to do work such as make ATP, expel toxins or concentrate useful molecules. Membrane spanning proteins have evolved to both generate and utilize the voltage across the membrane. Since protons are the most common ions that are transported across the membrane for these purposes, the measurement of the capacity for work is called the proton motive force. This project is focused on one protein called the transhydrogenase that uses the proton motive force to drive a chemical reaction to form a molecule, NADPH, that is essential for many cellular processes. For example, NADPH is needed for commercial microbial production of fermentation products, and manipulating the amount of transhydrogenase in the organism is one way to increase product yield. The project is aimed at determining the way in which a flux of protons flowing through the transhydrogenase protein from one side of the membrane (electrically positive) to the other (electrically negative) is coupled to driving a chemical reaction at a different location on the protein. The project will involve students at all levels; undergraduates, graduate students, including two women, and postdocs. The highly interdisciplinary scope of the research will allow students to be trained in a broad range of biochemical and biophysical techniques.This project is devoted to using biochemical and biophysical methods to examine the mechanism by which the membrane transhydrogenase couples hydride transfer from NADH to NADP+ to the proton electrochemical gradient across the membrane. The PI and his collaborators have determined the high-resolution structure of the protein that revealed that one domain of the 3-domain protein can occupy two dramatically different spatial orientations, more or less flipped by 180o. Based on this structure it can be postulated that in one configuration the substrate binds in a way that opens a proton channel through the membrane from one side only, allowing a histidine buried within the membrane to be protonated. The domain then "flips" and in the second configuration the product is formed by hydride transfer. The domain then flips back to the initial configuration in which the bound product induces the proton channel to open in the opposite direction, allowing proton dissociation. The model assumes large conformational changes during the catalytic cycle and also proposes a linkage between substrate binding and proton binding. These hypotheses will be experimentally tested using a combination of molecular biology, biochemistry and biophysical methodologies. NADPH binding to the enzyme will be tested using fluorescence anisotropy measurements. The enzyme will be reconstituted in proteoliposomes to allow the adjustment of the internal and external pH values along with the transmembrane voltage. Conformational dynamics will be examined by using distance-dependent spectroscopic methods using pairs of spin probes (PELDOR) and fluorescent probes (FRET) using modified cysteines placed at various regions in the protein.

所有生命的一个基本特征是从环境中提取能量并将其转化为有用的工作的能力。人类、其他动物、植物和微生物都有一套保存能量和将能量从一种形式转化为另一种形式的策略。这些策略中的一个核心要素是产生并利用隔膜上的电压。一种形式的能量被用来分离膜上的正电荷和负电荷，然后这个电压被用来驱动离子电流，离子电流被用来做功，比如制造ATP，排出毒素或浓缩有用的分子。跨膜蛋白已经进化为既能产生并利用跨膜电压。由于质子是为了这些目的而通过膜传输的最常见的离子，因此对功能力的测量称为质子推动力。这个项目的重点是一种名为转氢酶的蛋白质，它利用质子动力驱动化学反应形成NADPH分子，这是许多细胞过程中必不可少的。例如，商业微生物生产发酵产品需要NADPH，而控制生物体中转氢酶的数量是提高产品产量的一种方法。该项目旨在确定从膜的一侧(电正的)到另一侧(电负的)流经转氢酶蛋白质的质子通量是如何耦合到驱动蛋白质上不同位置的化学反应的。该项目将涉及所有层次的学生；本科生、研究生(包括两名女性)和博士后。这项研究的高度跨学科范围将允许学生接受广泛的生化和生物物理技术的培训。本项目致力于使用生化和生物物理方法来研究膜转氢酶将氢化物从NADH转移到NADP+到跨膜的质子电化学梯度的机制。PI和他的合作者已经确定了蛋白质的高分辨率结构，揭示了3-结构域蛋白质的一个结构域可以占据两个截然不同的空间方向，或多或少翻转了180度。基于这种结构，可以推测，在一种配置中，底物以仅从一侧打开穿过膜的质子通道的方式结合，允许掩埋在膜内的组氨酸被质子化。然后结构域“翻转”，在第二种构型中，产物是通过氢化物转移形成的。然后，结构域反转到初始构型，在初始构型中，结合产物诱导质子通道以相反的方向打开，从而允许质子解离。该模型假设在催化循环中有很大的构象变化，并提出了底物结合和质子结合之间的联系。这些假说将使用分子生物学、生物化学和生物物理方法的组合进行实验验证。将使用荧光各向异性测量来测试NADPH与酶的结合。该酶将在蛋白脂质体中重组，以允许内部和外部pH值随跨膜电压的变化而调整。构象动力学将通过使用自旋探针对(PELDOR)和荧光探针(FRET)的距离相关光谱方法来检测，这些探针使用位于蛋白质不同区域的修饰半胱氨酸。