Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, 405 30 Göteborg, Sweden.
Department of Chemistry and Molecular Biology, University of Gothenburg, 405 30 Göteborg, Sweden.
Biochemistry. 2020 May 5;59(17):1656-1660. doi: 10.1021/acs.biochem.9b01123. Epub 2020 Apr 6.
The cell envelope of Gram-negative bacteria is an elaborate cellular environment, consisting of two lipid membranes separated by the aqueous periplasm. So far, efforts to mimic this environment under laboratory conditions have been limited by the complexity of the asymmetric bacterial outer membrane. To evade this impasse, we recently established a method to modify the protein composition of bacterial outer membrane vesicles (OMVs) released from as a platform for biophysical studies of outer membrane proteins in their native membrane environment. Here, we apply protein-enriched OMVs to characterize the structure of three envelope proteins from using nuclear magnetic resonance (NMR) spectroscopy and expand the methodology to soluble periplasmic proteins. We obtain high-resolution NMR spectra of the transmembrane protein OmpA as well as the periplasmic proteins CpxP and MalE. We find that our approach facilitates structural investigations of membrane-attached protein domains and is especially suited for soluble proteins within their native periplasmic environment. Thereby, the use of OMVs in solution NMR methods allows analysis of the structure and dynamics of proteins twice the size compared to the current NMR methodology. We therefore expect our work to pave the way for more complex NMR studies of bacterial envelope proteins in the native environment of OMVs in the future.
革兰氏阴性菌的细胞包膜是一个精细的细胞环境，由两个脂质膜组成，中间隔着水相的周质空间。到目前为止，在实验室条件下模拟这种环境的努力受到不对称细菌外膜复杂性的限制。为了避开这个困境，我们最近建立了一种方法，从 中修饰细菌外膜囊泡 (OMV) 的蛋白质组成，将其作为在天然膜环境中研究外膜蛋白的生物物理特性的平台。在这里，我们应用富含蛋白质的 OMV 来表征三种包膜蛋白的结构，使用核磁共振 (NMR) 光谱法，并将该方法扩展到可溶性周质蛋白。我们获得了跨膜蛋白 OmpA 以及周质蛋白 CpxP 和 MalE 的高分辨率 NMR 谱。我们发现，我们的方法促进了膜结合蛋白结构域的结构研究，特别适合于其天然周质环境中的可溶性蛋白。因此，在溶液 NMR 方法中使用 OMV 可以分析比当前 NMR 方法大两倍的膜结合蛋白结构域的结构和动力学。因此，我们预计我们的工作将为未来在 OMV 的天然环境中对细菌包膜蛋白进行更复杂的 NMR 研究铺平道路。
