Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.
Institute of Chemical Sciences and Engineering, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.
Microbiologyopen. 2022 Jun;11(3):e1302. doi: 10.1002/mbo3.1302.
Gaining structural information on membrane proteins in their native lipid environment is a long-standing challenge in molecular biology. Instead, it is common to employ membrane mimetics, which has been shown to affect protein structure, dynamics, and function severely. Here, we describe the incorporation of a bacterial outer membrane protein (OmpW) into natively excreted membrane vesicles for solution nuclear magnetic resonance (NMR) spectroscopy using a mutant Escherichia coli strain with a high outer membrane vesicle (OMV) production rate. We collected NMR spectra from both vesicles containing overexpressed OmpW and vesicles from a control strain to account for the presence of physiologically relevant outer membrane proteins in vesicles and observed distinct resonance signals from OmpW. Due to the increased production of OMVs and the use of non-uniform sampling techniques we were able to obtain high-resolution 2D (HSQC) and 3D (HNCO) NMR spectra of our target protein inside its native lipid environment. While this workflow is not yet sufficient to achieve in situ structure determination, our results pave the way for further research on vesicle-based solution NMR spectroscopy.
在其天然脂质环境中获取膜蛋白的结构信息是分子生物学中长期存在的挑战。相反，通常采用膜类似物，这已被证明会严重影响蛋白质的结构、动力学和功能。在这里，我们描述了使用具有高外膜囊泡（OMV）产生率的突变大肠杆菌菌株，将一种细菌外膜蛋白（OmpW）掺入天然分泌的膜囊泡中，用于溶液核磁共振（NMR）光谱学。我们从含有过表达 OmpW 的囊泡和对照菌株的囊泡中收集 NMR 光谱，以说明囊泡中存在与生理相关的外膜蛋白，并观察到 OmpW 的独特共振信号。由于 OMV 的产量增加以及使用非均匀采样技术，我们能够在其天然脂质环境中获得目标蛋白的高分辨率 2D（HSQC）和 3D（HNCO）NMR 光谱。虽然该工作流程还不足以实现原位结构测定，但我们的结果为基于囊泡的溶液 NMR 光谱学的进一步研究铺平了道路。
