Abstract
Structural studies of membrane proteins (MP) in a native or native-like environment remain a challenge. X-ray crystallography of three-dimensional crystals of MP in lipids and cryo-electron microscopy of two-dimensional crystals also in lipids have given atomic structures of several MP. Recent developments of solid-state NMR (ssNMR) provided structural data of MP in lipids and should give access to the dynamic behavior of MP’s in a native-like environment. Preparation of samples for ssNMR is not trivial with overexpressed proteins since purified recombinant MP have to be reincorporated in proteoliposomes and concentrated in the small volume of the rotor used for ssNMR studies. We present here the protocol that we have used to study the recombinant mouse TSPO1, an integral membrane protein of 20 kDa mostly found in the outer membrane of mitochondria and overexpressed in E. coli bacteria.
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Acknowledgments
We would like to thank Stefanie Finet and Jean-Michel Guignier (UPMC, Paris) for their help and advice with DLS and cryo-EM experiments, Dominique Cailleu (Plateforme Analytique, Amiens) for help with NMR experiments on the 11.7 T magnet and Olivier Lequin (UPMC, Paris) for fruitful discussions.
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Senicourt, L., Duma, L., Papadopoulos, V., Lacapere, JJ. (2017). Solid-State NMR of Membrane Protein Reconstituted in Proteoliposomes, the Case of TSPO. In: Lacapere, JJ. (eds) Membrane Protein Structure and Function Characterization. Methods in Molecular Biology, vol 1635. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7151-0_18
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DOI: https://doi.org/10.1007/978-1-4939-7151-0_18
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