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 T₁₀₀ was used. The methodology to determine the orientation of membrane-reconstituted proteins from the accessibility of cysteines for thiol-specific reagents is critically evaluated.

• Purification;Reconstitution;Membrane Protein;Transport;Di-tripeptide