Manipulation of activity and orientation of membrane-reconstituted di-tripeptide transport protein DtpT of Lactococcus lactis

The di-tripeptide transport system (DtpT) of Lactococcus lactis was purifie d to apparent homogeneity by pre-extraction of crude membrane vesicles with octaethylene glycol monodecyl ether (C10E8), followed by solubilization wi th n-dodecyl-beta-D-maltoside (DDM) and chromatography on a Ni-NTA resin. T he DtpT protein was reconstituted into detergent-destabilized preformed lip osomes prepared from E. coli phospholipid/phosphatidylcholine. A variety of detergents were tested for their ability to mediate the membrane reconstit ution 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 solubilizatio n of the preformed liposomes. Parallel with the low activity, significant l osses of lipid were observed when the reconstitution was performed at high DDM concentrations. This explained at least part of the reduced transport a ctivity as the DtpT protein was highly dependent on the final lipid-to-prot ein ratios in the proteoliposomes. Consistent with the difference in mechan ism of DDM- and TX100-mediated membrane protein reconstitution, the orienta tion 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 membra ne-reconstituted proteins from the accessibility of cysteines for thiol-spe cific reagents is critically evaluated.