Abstract
The study of membrane proteins is a difficult task due to their natural embedding in hydrophobic environment made by lipids. Solubilization and purification from native membranes or overexpressed system involves the use of detergent to make them soluble while maintaining their structural and functional properties. The choice of detergent is governed not only by their ability to reach these goals, but also by their compatibility with biochemical and structural studies. A different detergent can be used during purification, and characterization of the detergent amounts present in each purification step is crucial. To address this point, we developed a colorimetric method to measure detergent content in different preparations. We analyzed detergent present in the collected fractions from the purification of the recombinant membrane translocator protein (RecTSPO). We followed detergent removal during the reconstitution of RecTSPO in liposomes and observed by electron microscopy the formation of proteoliposomes. We addressed the RecTSPO functionality by testing its ability to bind high affinity drug ligand [3H]PK 11195. We described the different parameters that should be controlled in order to optimize the measurement of this ligand binding using a filtration procedure. These protocols are useful to characterize functionality and detergent content of membrane protein, both key factors for further structural studies.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Lacapere J-J, Pebay-Peyroula E, Neumann J-M, Etchebest C (2007) Determining membrane protein structures: still a challenge! Trends Biochem Sci 32:259–270
Special issue of BBA biomembrane 1610:1–153
Garavito RM, Ferguson-Miller S (2001) Detergents as tools in membrane biochemistry. J Biol Chem 276:32403–32406
LeMaire M, Champeil P, Møller JV (2000) Interaction of membrane proteins and lipids with solubilizing detergents. Biochim Biophys Acta 1508:86–111
Pebay-Peyroula E (ed) (2008) Biophysical analysis of membrane protein. Investigating structure and function. Wiley-VCH, Weinheim
Gasteiger E, Gattiker A, Hoogland C, Ivanyi I, Appel RD, Bairoch A (2003) ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res 31:3784–3788
Fleischer S, Rouser G, Fleischer B, Casu A, Kritchevski G (1967) Lipid composition of mitochondria from bovin heart, liver, and kidney. J Lipid Res 8:170–180
Schiller J, Arnhold J, Benard S, Müller M, Reichl S, Arnold K (1999) Lipid analysis by matrix-assisted laser desorption and ionization mass spectroscopy: a methodological approach. Anal Biochem 267:46–56
Mokus M, Kragh-Hansen U, Letellier P, le Maire M, Møller JV (1998) Construction and use of a detergent-sensitive electrode to measure dodecyl sulfate activity and binding. Anal Biochem 264:34–40
Eriks LR, Mayor JA, Kaplan RS (2003) A strategy for identification and quantification of detergents frequently used in the purification of membrane proteins. Anal Biochem 323:234–241
Papadopoulos V, Baraldi M, Guilarte TR, Knudsen TB, Lacapere J-J, Lindemann P, Noremberg MD, Nutt D, Weizman A, Zhang M-R, Gavish M (2006) Translocator protein (18 kDa): new nomenclature for the peripheral-type benzodiazepine receptor based on its structure and molecular function. Trends Pharmacol Sci 27:402–409
Holloway PW (1973) A simple procedure for removal of triton X-100 from proteins samples. Anal Biochem 53:304–340
Rigaud J-L, Mosser G, Lacapere J-J, Olofson A, Levy D, Ranck J-L (1998) Bio-Beads: an efficient strategy for two-dimensional crystallization of membrane proteins. J Struct Biol 118:226–235
Lacapere J-J, Stokes DL, Olofsson A, Rigaud J-L (1998) Two-dimensional crystallization of Ca-ATPase by detergent removal. Biophys J 75:1319–1329
Lacapere J-J, Delavoie F, Li H, Péranzi G, Maccario J, Papadopulos V, Vidic B (2001) Structural and functional study of reconstituted peripheral benzodiazepine receptor. Biochem Biophys Res Commun 284:536–541
Rigaud J-L, Pitard B, Levy D (1995) Reconstitution of membrane proteins into liposomes: application to energy-transducing membrane proteins. Biochim Biophys Acta 1231:223–246
Acknowledgment
The authors would like to thank Professor V. Papadopoulos for the generous gift of TSPO plasmids, G. Péranzi and A. Letort for their contribution in preliminary experiments. This work was supported by CNRS (Centre National de la Recherche Scientifique) and ANR (Agence National pour la Recherche) Grant 06-Blan-0190-01 to JJL.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Ostuni, M.A., Iatmanen, S., Teboul, D., Robert, JC., Lacapère, JJ. (2010). Characterization of Membrane Protein Preparations: Measurement of Detergent Content and Ligand Binding After Proteoliposomes Reconstitution. In: Lacapère, JJ. (eds) Membrane Protein Structure Determination. Methods in Molecular Biology, vol 654. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-762-4_1
Download citation
DOI: https://doi.org/10.1007/978-1-60761-762-4_1
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-761-7
Online ISBN: 978-1-60761-762-4
eBook Packages: Springer Protocols