Institute of Biophysics, Department of Physics, Goethe University Frankfurt, Frankfurt/Main, Germany.
Department of Physics, Freie Universität Berlin, Berlin, Germany.
Methods Mol Biol. 2024;2778:237-257. doi: 10.1007/978-1-0716-3734-0_15.
Outer membrane proteins (OMPs) of Gram-negative bacteria are involved in many essential functions of the cell. They are tightly packed in the outer membrane, which is an asymmetric lipid bilayer. Electron spin resonance (ESR) spectroscopic techniques combined with site-directed spin labeling (SDSL) enable observation of structure and conformational dynamics of these proteins directly in their native environments. Here we depict a protocol for site-directed spin labeling of β-barrel membrane proteins in isolated outer membranes and intact E. coli using nitroxide, triarylmethyl (trityl), and Gd-based spin tags. Furthermore, subsequent continuous wave (CW) and orthogonal pulsed electron-electron double resonance (PELDOR) measurements are described along with experimental setup at Q-band (34 GHz), the data analysis, and interpretation.
革兰氏阴性细菌的外膜蛋白（OMPs）参与细胞的许多基本功能。它们紧密地排列在外膜中，外膜是一个不对称的脂质双层。电子自旋共振（ESR）光谱技术与定点自旋标记（SDSL）相结合，可以直接在天然环境中观察这些蛋白质的结构和构象动力学。在这里，我们描述了一种使用氮氧自由基、三芳基甲基（三苯甲基）和基于 Gd 的自旋标记物在分离的外膜和完整的大肠杆菌中定点标记β桶膜蛋白的方案。此外，还描述了随后的连续波（CW）和正交脉冲电子-电子双共振（PELDOR）测量，以及在 Q 波段（34 GHz）的实验设置、数据分析和解释。
