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Sample Preparation of Rhodopsins in the E. coli Membrane for In Situ Magic Angle Spinning Solid-State Nuclear Magnetic Resonance Studies

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Chemical and Synthetic Approaches in Membrane Biology

Part of the book series: Springer Protocols Handbooks ((SPH))

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Abstract

Determination of the structure and dynamics of membrane proteins in complex, native cellular environments is one of the primary targets of structural biology. Here, we present a protocol for the preparation of recombinant membrane proteins in the native E. coli membrane environment for solid-state NMR (SSNMR) studies. This protocol has been developed on Anabaena sensory rhodopsin (ASR), a seven-transmembrane α-helical light receptor, but should be easily transferable to similar recombinant membrane protein systems. In order for SSNMR studies to be possible on such complex systems, it is desirable to remove as much background signal as possible. This is achieved both through physically separating segments of the membrane containing ASR and through isotopic labeling strategies which strategically limit isotopic incorporation into background proteins. Through the implementation of these methods and 3D SSNMR spectroscopy, we find that it is possible to resolve and characterize 40% of the previously assigned residues of ASR in the E. coli membrane environment.

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Abbreviations

ASR:

Anabaena Sensory Rhodopsin

EM:

E. coli membrane

IM:

Inner membrane

MAS:

Magic angle spinning

NA:

Natural abundance

NIC:

Non-induced cells

NMR:

Nuclear magnetic resonance

OM:

Outer membrane

rbUCN/rbUN:

Reduced-background UCN/UN labeled

SSNMR:

Solid-state NMR

UCN:

Uniformly 13C,15N-labeled

UN:

Uniformly 15N-labeled

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Acknowledgments

We thank Ms. Emily Ritz for providing us with the UCN PL-ASR sample and Ms. Rachel Munro for her preliminary work on the isolation procedure. This research was supported by the Natural Sciences and Engineering Research Council of Canada (Discovery Grants to V.L. and L.S.B.). MEW is a recipient of the Ontario Graduate Scholarship.

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Authors and Affiliations

  1. Department of Physics and Biophysics Interdepartmental Group, University of Guelph, Guelph, ON, Canada, N1G 2W1

    Meaghan E. Ward, Vladimir Ladizhansky & Leonid S. Brown

Authors
  1. Meaghan E. Ward
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  2. Vladimir Ladizhansky
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  3. Leonid S. Brown
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Corresponding authors

Correspondence to Vladimir Ladizhansky or Leonid S. Brown .

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Editors and Affiliations

  1. Indian Institute of Technology, Kanpur, India

    Arun K. Shukla

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Ward, M.E., Ladizhansky, V., Brown, L.S. (2016). Sample Preparation of Rhodopsins in the E. coli Membrane for In Situ Magic Angle Spinning Solid-State Nuclear Magnetic Resonance Studies. In: Shukla, A. (eds) Chemical and Synthetic Approaches in Membrane Biology. Springer Protocols Handbooks. Humana Press, New York, NY. https://doi.org/10.1007/8623_2016_5

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  • DOI: https://doi.org/10.1007/8623_2016_5

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6835-0

  • Online ISBN: 978-1-4939-6836-7

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