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Lipid bilayer preparations of membrane proteins for oriented and magic-angle spinning solid-state NMR samples

Nature Protocols volume 8, pages 2256–2270 (2013)Cite this article

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Abstract

Solid-state NMR spectroscopy has been used successfully for characterizing the structure and dynamics of membrane proteins as well as their interactions with other proteins in lipid bilayers. Such an environment is often necessary for achieving native-like structures. Sample preparation is the key to this success. Here we present a detailed description of a robust protocol that results in high-quality membrane protein samples for both magic-angle spinning and oriented-sample solid-state NMR. The procedure is demonstrated using two proteins: CrgA (two transmembrane helices) and Rv1861 (three transmembrane helices), both from Mycobacterium tuberculosis. The success of this procedure relies on two points. First, for samples for both types of NMR experiment, the reconstitution of the protein from a detergent environment to an environment in which it is incorporated into liposomes results in 'complete' removal of detergent. Second, for the oriented samples, proper dehydration followed by rehydration of the proteoliposomes is essential. By using this protocol, proteoliposome samples for magic-angle spinning NMR and uniformly aligned samples (orientational mosaicity of <1°) for oriented-sample NMR can be obtained within 10 d.

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Figure 1: Schematic of the experimental procedure with expected timeline from membrane protein expression to NMR spectroscopy.
Figure 2: Reconstitution of membrane proteins by MβCD.
Figure 3: Detailed procedure for mechanically aligned glass slide–supported OS ssNMR sample preparation of membrane proteins.
Figure 4: Step-by-step procedure for MAS sample transfer to a thin-walled 3.2-mm MAS rotor.
Figure 5: SDS-PAGE (12%) gels illustrating incomplete and good reconstitution.
Figure 6: ssNMR 31P and 15N 1D spectra of uniformly 15N-labeled gramicidin A, CrgA and Rv1861 in lipid bilayers.
Figure 7: Two-dimensional separated local field (SLF) ssNMR spectra of CrgA and Rv1861 in oriented lipid bilayers.
Figure 8: Two-dimensional 13C-13C DARR-MAS spectra of CrgA and Rv1861 in liposomal preparations at 243 K with a 10-kHz spinning rate and 30-ms mixing time.

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Acknowledgements

We thank M.W. Davidson at NHMFL and C. Escobar at FSU, IMB, NHMFL for helping with photography. We also thank P.L. Gor'kov for his design of the OS sample holder and the sample transfer base. This work was supported in part by the US National Institutes of Health (grants AI 074805, AI 073891 and AI 023007) and the US National Science Foundation (through Cooperative Agreement 0654118 between the Division of Materials Research and the State of Florida).

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Authors and Affiliations

  1. Institute of Molecular Biophysics (IMB), Florida State University (FSU), Tallahassee, Florida, USA

    Nabanita Das, Dylan T Murray & Timothy A Cross

  2. National High Magnetic Field Laboratory (NMHFL), FSU, Tallahassee, Florida, USA

    Nabanita Das, Dylan T Murray & Timothy A Cross

  3. Department of Chemistry and Biochemistry, FSU, Tallahassee, Florida, USA

    Timothy A Cross

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  1. Nabanita Das
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Contributions

N.D. and D.T.M. performed all the experiments such as membrane protein expression, purification, solid-state NMR sample preparation and new method development. N.D. prepared all the figures. N.D. and T.A.C. wrote the manuscript, D.T.M. provided essential comments. All three authors coordinated to complete this manuscript.

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Correspondence to Timothy A Cross.

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Integrated supplementary information

Supplementary Figure 1 12% (wt/vol) SDS-PAGE of CrgA and Rv1861 membrane protein expression and purification steps.

M: Molecular weight marker, L: Whole cell lysate containing inclusion body and membrane fractions, FT: Flow through from nickel column, Washes: two to three consecutive washing steps, Elutions: Protein elution from nickel column. Molecular weights of the proteins are shown by red color arrows.

Supplementary information

Supplementary Figure 1

12% (wt/vol) SDS-PAGE of CrgA and Rv1861 membrane protein expression and purification steps. (PDF 3068 kb)

Supplementary Methods

CrgA and Rv1861 membrane protein expression and purification; Reconstitution and OS sample preparation of 15N uniform labeled gramicidin A protein in DMPC lipid bilayers. (PDF 208 kb)

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Das, N., Murray, D. & Cross, T. Lipid bilayer preparations of membrane proteins for oriented and magic-angle spinning solid-state NMR samples. Nat Protoc 8, 2256–2270 (2013). https://doi.org/10.1038/nprot.2013.129

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