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Electron paramagnetic resonance (EPR) spectroscopy of spin-labeled membrane proteins is a valuable biophysical technique to study structural details and conformational transitions of proteins close to their physiological environment, e.g., in liposomes, membrane bilayers, and nanodiscs. Unlike in nuclear magnetic resonance (NMR) spectroscopy, having only one or a few specific side chains labeled at a time with paramagnetic probes makes the size of the object under investigation irrelevant in terms of technique sensitivity. As a drawback, extensive site-directed mutagenesis is required in order to analyze the properties of the protein under investigation. EPR can provide detailed information on side chain dynamics of large membrane proteins or protein complexes embedded in membranes with exquisite sensitivity for flexible regions and on water accessibility profiles across the membrane bilayer. Moreover, distances between the two spin-labeled side chains in membrane proteins can be detected with high precision at cryogenic temperatures. The application of EPR to membrane proteins still presents some challenges in terms of sample preparation, sensitivity, and data interpretation; thus, it is difficult to give ready-to-go methodological recipes. However, new technological developments (arbitrary waveform generators) and new spin labels spectroscopically orthogonal to nitroxides increased the range of applicability from in vitro toward in cell EPR experiments. This chapter is an updated version of the one published in the first edition of the book, and describes the state of the art in the application of nitroxide-based site-directed spin labeling EPR to membrane proteins, addressing new tools such as arbitrary waveform generators and spectroscopically orthogonal labels, such as Gd(III)-based labels. We will present challenges in sample preparation and data analysis for functional and structural membrane protein studies using site-directed spin labeling techniques and give experimental details on EPR techniques, providing information on side chain dynamics and water accessibility using nitroxide probes. An optimal Q-band DEER setup for nitroxide probes will be described, and its extension to gadolinium-containing samples will be addressed.

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EPR Techniques to Probe Insertion and Conformation of Spin-Labeled Proteins in Lipid Bilayers

  • 实验方案

  • 发表日期：

    2026年

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  • Enrica Bordignon,
  • Svetlana Kucher,
  • Yevhen Polyhach

摘要

Electron paramagnetic resonance (EPR) spectroscopy of spin-labeled membrane proteins is a valuable biophysical technique to study structural details and conformational transitions of proteins close to their physiological environment, e.g., in liposomes, membrane bilayers, and nanodiscs. Unlike in nuclear magnetic resonance (NMR) spectroscopy, having only one or a few specific side chains labeled at a time with paramagnetic probes makes the size of the object under investigation irrelevant in terms of technique sensitivity. As a drawback, extensive site-directed mutagenesis is required in order to analyze the properties of the protein under investigation. EPR can provide detailed information on side chain dynamics of large membrane proteins or protein complexes embedded in membranes with exquisite sensitivity for flexible regions and on water accessibility profiles across the membrane bilayer. Moreover, distances between the two spin-labeled side chains in membrane proteins can be detected with high precision at cryogenic temperatures. The application of EPR to membrane proteins still presents some challenges in terms of sample preparation, sensitivity, and data interpretation; thus, it is difficult to give ready-to-go methodological recipes. However, new technological developments (arbitrary waveform generators) and new spin labels spectroscopically orthogonal to nitroxides increased the range of applicability from in vitro toward in cell EPR experiments. This chapter is an updated version of the one published in the first edition of the book, and describes the state of the art in the application of nitroxide-based site-directed spin labeling EPR to membrane proteins, addressing new tools such as arbitrary waveform generators and spectroscopically orthogonal labels, such as Gd(III)-based labels. We will present challenges in sample preparation and data analysis for functional and structural membrane protein studies using site-directed spin labeling techniques and give experimental details on EPR techniques, providing information on side chain dynamics and water accessibility using nitroxide probes. An optimal Q-band DEER setup for nitroxide probes will be described, and its extension to gadolinium-containing samples will be addressed.