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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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).
The authors declare no competing financial interests.
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.
12% (wt/vol) SDS-PAGE of CrgA and Rv1861 membrane protein expression and purification steps. (PDF 3068 kb)
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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