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

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Transmembrane β-Barrel Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2778))

Abstract

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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Acknowledgments

This work was financially supported from the Deutsche Forschungsgemeinschaft via the Emmy Noether program (JO 1428/1−1), SFB 1507 − “Membrane-associated Protein Assemblies, Machineries, and Supercomplexes” and a large equipment fund (438280639) to B.J. Trityl labels were provided by Victor M. Tormyshev and Elena G. Bagryanskaya.

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Author notes

  1. Authors Sophie Ketter and Aathira Gopinath have equally contributed to this chapter.

Authors and Affiliations

  1. Institute of Biophysics, Department of Physics, Goethe University Frankfurt, Frankfurt/Main, Germany

    Sophie Ketter & Aathira Gopinath

  2. Department of Physics, Freie Universität Berlin, Berlin, Germany

    Aathira Gopinath & Benesh Joseph

Authors
  1. Sophie Ketter
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  2. Aathira Gopinath
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  3. Benesh Joseph
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Corresponding author

Correspondence to Benesh Joseph .

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Editors and Affiliations

  1. Laboratoire de Microbiologie et Génétique Moléculaires, Centre de Biologie Intégrative, CNRS, Université de Toulouse, Toulouse, France

    Raffaele Ieva

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Ketter, S., Gopinath, A., Joseph, B. (2024). Conformational Heterogeneity of β-Barrel Membrane Proteins Observed In Situ Using Orthogonal Spin Labels and Pulsed ESR Spectroscopy. In: Ieva, R. (eds) Transmembrane β-Barrel Proteins. Methods in Molecular Biology, vol 2778. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3734-0_15

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  • DOI: https://doi.org/10.1007/978-1-0716-3734-0_15

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3733-3

  • Online ISBN: 978-1-0716-3734-0

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