利用正交自旋标签和脉冲 ESR 光谱原位观测 β 管膜蛋白的构象异质性

Q4 Biochemistry, Genetics and Molecular Biology Methods in molecular biology Pub Date : 2024-01-01 DOI:10.1007/978-1-0716-3734-0_15

Sophie Ketter, Aathira Gopinath, Benesh Joseph

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引用次数: 0

摘要

革兰氏阴性细菌的外膜蛋白（OMPs）参与了细胞的许多基本功能。它们紧紧包裹在不对称脂质双分子层的外膜中。电子自旋共振（ESR）光谱技术与位点定向自旋标记（SDSL）相结合，可直接观察这些蛋白质在其原生环境中的结构和构象动态。在这里，我们描述了一种利用硝基氧化物、三芳甲基（triarylmethyl）和基于 Gd3+ 的自旋标签对离体外膜和完整大肠杆菌中的β管膜蛋白进行位点定向自旋标记的方案。此外，还介绍了随后的连续波（CW）和正交脉冲电子-电子双共振（PELDOR）测量，以及 Q 波段（34 GHz）的实验设置、数据分析和解释。

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Conformational Heterogeneity of β-Barrel Membrane Proteins Observed In Situ Using Orthogonal Spin Labels and Pulsed ESR Spectroscopy.

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 Gd³⁺-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.

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来源期刊

Methods in molecular biology Biochemistry, Genetics and Molecular Biology-Genetics

CiteScore

2.00

自引率

0.00%

发文量

3536

期刊介绍： For over 20 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice.