Characterization of membrane protein preparations: measurement of detergent content and ligand binding after proteoliposomes reconstitution.
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 [(3)H]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.
Ostuni
Mariano A
MA
INSERM U773, Centre de Recherche Biomédicale Bichat-Beaujon (CRB3), Faculté de Médecine X, Bichat, Université Paris 7, Paris, France. maraino.ostuni@inserm.fr
Iatmanen
Soria
S
Teboul
David
D
Robert
Jean-Claude
JC
Lacapère
Jean-Jacques
JJ
D003902:Detergents / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000737:chemistry; D011510:Proteolipids / Q000737:chemistry*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry
D003902:Detergents; D008565:Membrane Proteins; D011510:Proteolipids; C020485:proteoliposomes; D010767:Phosphorylcholine; D012967:Sodium Dodecyl Sulfate; C028810:dodecylphosphocholine