Q-Band Pulsed EPR Spectrometer

我们的团队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，这种膜蛋白实验非常具有挑战性。低表达水平往往导致样本数量少。此外，当自旋标签附着在膜蛋白上时，其相记忆时间显著缩短。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，也使较长距离（bbb50 nm）的测量变得极其困难。在膜蛋白结构生物学中，越来越多的证据表明天然脂质环境对蛋白质的折叠、结构和功能起着至关重要的作用。本课课组的主要工作之一是开发和应用脉冲电子-电子双共振原位研究膜蛋白。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，这更具挑战性。在过去的几年里，高性能脉冲光谱仪的出现大大提高了膜蛋白实验的灵敏度。该装置配备了一个高功率微波放大器和一个任意波形发生器，它们一起为膜蛋白的新研究提供了前所未有的机会。利用这种装置，我们的目标是表征不同膜转运蛋白复合物的构象异质性和平衡动力学，这些构象异质性和平衡动力学构成了功能的基础。新方法，包括不同的自旋标签，标记策略，以及原位电子自旋共振光谱的样品制备方案将进行测试。随后，这些方法将应用于研究蛋白质折叠-桶组装机械复合物和革兰氏阴性菌的脂多糖运输系统。这两种系统在革兰氏阴性菌中都是必需的和保守的，因此是新药非常受欢迎的靶点。此外，我们一直在研究初级和次级活性膜转运蛋白的底物转运机制。利用ATP结合盒输出器TmrAB，我们证明了在三个单独的结构域独立观察构象平衡的可行性。利用质子偶联富马酸同体SLC26Dg，利用脉冲电子-电子双共振约束确定了蛋白质脂质体的二聚体结构。我们的目标是进一步阐明蛋白质-底物和蛋白质-脂质相互作用的细节，并探索这些转运体在底物易位过程中热力学参数的变化。

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.