Detergent-mediated reconstitution of membrane proteins.
The efficiency of reconstitution of the lactose transport protein (LacS) of Streptococcus thermophilus is markedly higher with Triton X-100 than with other detergents commonly employed to mediate the membrane insertion. To rationalize these differences, the lipid/detergent structures that are formed in the reconstitution process were studied by cryotransmission electron microscopy. Surprisingly, the two nonionic detergents Triton X-100 and n-dodecyl beta-D-maltoside (DDM) affected the liposome structures in a completely different manner. Preformed liposomes titrated with Triton X-100 maintained their bilayer structure far beyond the onset of solubilization, and transport activity was maximal when LacS was inserted into these structures. With DDM the vesicular structures were already disrupted at the onset of solubilization and these membrane sheets were converted into long threadlike micelles at higher DDM to lipid ratios. Triton X-100 allowed the protein to be reconstituted with the hydrophilic surface exposed to the outside, whereas LacS was incorporated randomly when DDM was used. These differences in orientation are readily explained by the different lipid-detergent structures formed by Triton X-100 and DDM. The orientation of the reconstituted LacS protein is a critical factor for the activity of the protein as the kinetics of translocation is very different for opposite directions of transport.
Knol
J
J
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.
Sjollema
K
K
Poolman
B
B
D001426:Bacterial Proteins / Q000378:metabolism* / Q000648:ultrastructure; D001692:Biological Transport; D003902:Detergents / Q000494:pharmacology*; D029968:Escherichia coli Proteins; D005960:Glucosides / Q000494:pharmacology; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism / Q000648:ultrastructure; D008565:Membrane Proteins / Q000378:metabolism* / Q000648:ultrastructure; D026901:Membrane Transport Proteins / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008954:Models, Biological; D008956:Models, Chemical; D009004:Monosaccharide Transport Proteins; D017830:Octoxynol / Q000494:pharmacology; D011510:Proteolipids / Q000378:metabolism; D013291:Streptococcus / Q000201:enzymology; D027981:Symporters; D017186:Titrimetry
D001426:Bacterial Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; D005960:Glucosides; C050221:LacY protein, E coli; D008081:Liposomes; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011510:Proteolipids; D027981:Symporters; C020485:proteoliposomes; C040358:dodecyl maltoside; D017830:Octoxynol; C021499:lactose permease