Q-Band Pulsed EPR Spectrometer

Q 波段脉冲 EPR 光谱仪

• 批准号: 438280639
• 金额: --
• 依托单位: Goethe-Universität Frankfurt am Main
• 依托单位国家: 德国
• 项目类别: Major Research Instrumentation
• 财政年份: 2020
• 资助国家: 德国
• 起止时间: 2019-12-31 至 无数据
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/438280639?language=en
• 关键词: Band; Pulsed; EPR; Spectrometer

Our group investigates the structure, function, and dynamics of a few important membrane protein complexes. Electron spin resonance spectroscopy techniques, especially pulsed electron-electron double resonance is used as the major biophysical tool in our research. Such experiments on membrane proteins are very challenging for several reasons. The low expression levels often lead to small sample quantities. In addition, when attached to membrane proteins, the phase memory time of the spin labels get significantly reduced. These limitations combined with the narrow excitation bandwidth of conventional rectangular microwave pulses lead to poor sensitivity and also make the measurement of longer distances (> 5 nm) extremely difficult. For membrane protein structural biology, there is increasing evidence for the vital role of native lipid environment for protein folding, structure, and function. One of the major activities of our working group is to develop and apply pulsed electron-electron double resonance for studying membrane proteins in situ. For a few additional reasons such as the background labeling, instability of the spin labels, and the low protein expression, etc., this is even more challenging. Over the past few years, the availability of a high-performance pulsed spectrometer has greatly enhanced the sensitivity for such experiments with membrane proteins. This device is equipped with a high-power microwave amplifier and an arbitrary waveform generator, which together offer unprecedented opportunities for novel investigations in membrane proteins. Using such a device, we aim to characterize the conformational heterogeneity and the equilibrium dynamics that form the basis of function in different membrane transport protein complexes. New approaches, including different spin labels, labeling strategies, and sample preparation protocols for in situ electron spin resonance spectroscopy will be tested. Later those approached will be applied to study the protein folding beta-barrel assembly machinery complex and the lipopolysaccharide transport systems of Gram-negative bacteria. Both of these systems are essential and conserved in Gram-negative bacteria and therefore are highly sought-after targets for new drugs. In addition, we have been studying the substrate translocation mechanism for primary and secondary active membrane transporters. With the ATP Binding Cassette exporter TmrAB, we showed the feasibility to independently observe the conformational equilibria at three individual domains. With the proton-coupled fumarate symporter SLC26Dg, we determined the dimer structure in proteoliposomes using pulsed electron-electron double resonance constraints. We aim to further elucidate the details of protein-substrate and protein-lipid interactions as well to explore the changes in the thermodynamic parameters during substrate translocation in these transporters.

我们小组研究了一些重要的膜蛋白复合物的结构、功能和动力学。电子自旋共振波谱技术，特别是脉冲电子-电子双共振波谱技术是我们研究的主要生物物理工具。由于几个原因，这种膜蛋白的实验非常具有挑战性。低表达水平通常导致小的样品量。此外，当连接到膜蛋白时，自旋标记的相位记忆时间显着减少。这些限制与传统矩形微波脉冲的窄激发带宽相结合导致灵敏度差，并且还使得测量较长距离（> 5 nm）极其困难。对于膜蛋白结构生物学，有越来越多的证据表明天然脂质环境对蛋白质折叠，结构和功能的重要作用。我们工作组的主要活动之一是发展和应用脉冲电子-电子双共振技术原位研究膜蛋白。由于一些额外的原因，如背景标记、自旋标记的不稳定性和低蛋白表达等，这就更具挑战性了。在过去的几年里，高性能脉冲光谱仪的可用性大大提高了膜蛋白实验的灵敏度。该设备配备了高功率微波放大器和任意波形发生器，它们共同为膜蛋白的新研究提供了前所未有的机会。使用这样的设备，我们的目标是表征的构象异质性和平衡动力学，形成在不同的膜转运蛋白复合物的功能的基础。新的方法，包括不同的自旋标记，标记策略，并在原位电子自旋共振光谱样品制备协议将进行测试。这些方法将用于研究革兰氏阴性菌的蛋白质折叠、β-桶组装机制复合体和脂多糖转运系统。这两个系统在革兰氏阴性菌中是必需的和保守的，因此是新药的高度抢手的靶标。此外，我们还研究了初级和次级活性膜转运蛋白的底物转运机制。与ATP结合盒出口TmrAB，我们表明独立观察在三个单独的域的构象平衡的可行性。利用质子偶联的富马酸同向转运体SLC 26 Dg，我们利用脉冲电子-电子双共振约束确定了脂蛋白体中的二聚体结构。我们的目的是进一步阐明蛋白质-底物和蛋白质-脂质相互作用的细节，以及探索在这些转运蛋白的底物易位过程中的热力学参数的变化。