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Bacterial Production and Solution NMR Studies of a Viral Membrane Ion Channel

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Protein NMR Techniques

Part of the book series: Methods in Molecular Biology ((MIMB,volume 831))

Abstract

Advances in solution nuclear magnetic resonance (NMR) methodology that enable studies of very large proteins have also paved the way for studies of membrane proteins that behave like large proteins due to the added weight of surfactants. Solution NMR has been used to determine the high-resolution structures of several small, membrane proteins dissolved in detergent micelles and small bicelles. However, the usual difficulties with membrane proteins in producing, purifying, and stabilizing the proteins away from native membranes remain, requiring intensive screening efforts. Low levels of heterologous expression can be the most detrimental aspect to studying membrane proteins. This is exacerbated for NMR studies because of the costs of isotopically enriched media. Thus, solution NMR studies have tended to focus on relatively small, membrane proteins that can be expressed into inclusion bodies and refolded. Here, we describe the methods used to produce, purify, and refold the proton channel M2 into detergent micelles, and the procedures used to determine chemical shift assignments and the atomic level structure of the closed form of the homotetrameric channel.

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Acknowledgments

James J. Chou and the National Institutes of Health, USA (NIH) are acknowledged for supporting development of the approaches described. J.R.S. was supported by a Ruth Kirschstein Fellowship from the NIH. Matthew E. Call is acknowledged for many useful modifications and additions to the trpLE fusion expression and purification protocol.

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Authors and Affiliations

  1. Department of Biochemistry, University of Oxford, Oxford, UK

    Jolyon K. Claridge & Jason R. Schnell

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  1. Jolyon K. Claridge
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  2. Jason R. Schnell
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Correspondence to Jason R. Schnell .

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Editors and Affiliations

  1. University of Albany, State University of New York, Washington Avenue 1400, Albany, 12222, New York, USA

    Alexander Shekhtman

  2. University of Albany, State University of New York, Washington Avenue 1400, Albany, 12222, New York, USA

    David S. Burz

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Claridge, J.K., Schnell, J.R. (2012). Bacterial Production and Solution NMR Studies of a Viral Membrane Ion Channel. In: Shekhtman, A., Burz, D. (eds) Protein NMR Techniques. Methods in Molecular Biology, vol 831. Humana Press. https://doi.org/10.1007/978-1-61779-480-3_10

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  • DOI: https://doi.org/10.1007/978-1-61779-480-3_10

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  • Publisher Name: Humana Press

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  • Online ISBN: 978-1-61779-480-3

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