title: Manipulation of activity and orientation of membrane-reconstituted di-tripeptide transport protein DtpT of Lactococcus lactis.
abstract: The di-tripeptide transport system (DtpT) of Lactococcus lactis was purified to apparent homogeneity by pre-extraction of crude membrane vesicles with octaethylene glycol monodecyl ether (C10E8), followed by solubilization with n-dodecyl-beta-D-maltoside (DDM) and chromatography on a Ni-NTA resin. The DtpT protein was reconstituted into detergent-destabilized preformed liposomes prepared from E. coli phospholipid/phosphatidylcholine. A variety of detergents were tested for their ability to mediate the membrane reconstitution of DtpT and their effectiveness to yield proteoliposomes with a high transport activity. The highest activities were obtained with TX100, C12E8 and DM, whereas DDM yielded relatively poor activities, in particular when this detergent was used at concentrations beyond the onset of solubilization of the preformed liposomes. Parallel with the low activity, significant losses of lipid were observed when the reconstitution was performed at high DDM concentrations. This explained at least part of the reduced transport activity as the DtpT protein was highly dependent on the final lipid-to-protein ratios in the proteoliposomes. Consistent with the difference in mechanism of DDM- and TX100-mediated membrane protein reconstitution, the orientation of the DtpT protein in the membrane was random with DDM and inside-in when TX100 was used. The methodology to determine the orientation of membrane-reconstituted proteins from the accessibility of cysteines for thiol-specific reagents is critically evaluated.
authors.affiliation: Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.
authors.forename: G
authors.initials: G
authors.lastname: Fang
authors.forename: R
authors.initials: R
authors.lastname: Friesen
authors.forename: F
authors.initials: F
authors.lastname: Lanfermeijer
authors.forename: A
authors.initials: A
authors.lastname: Hagting
authors.forename: B
authors.initials: B
authors.lastname: Poolman
authors.forename: W N
authors.initials: WN
authors.lastname: Konings
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002499:Centrifugation, Density Gradient; D003902:Detergents; D004151:Dipeptides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005960:Glucosides; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013294:Lactococcus lactis / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry; D008563:Membrane Lipids / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D026901:Membrane Transport Proteins; D011092:Polyethylene Glycols
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D003902:Detergents; D004151:Dipeptides; D005960:Glucosides; D008081:Liposomes; D008563:Membrane Lipids; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C038123:alanylproline; C086845:dtpT protein, Lactococcus lactis; C017272:dodecyloctaethyleneglycol monoether; D011092:Polyethylene Glycols; C040358:dodecyl maltoside