Principal Investigator

We try to get more structural, functional and mechanistic information on secondary active transporters in a systematic approach. We have selected 42 protein families of secondary active transporters comprising around 270 individual transporters from Aquifex aeolicus, Pyrococcus furiosus, Salmonella typhimurium and humans. The genes coding for these transporters are expressed in Escherichia coli as fusion proteins with affinity tags using a set of six different vectors. Crystals of eight different secondary transporters have been obtained so far. We have already determined the structure of the sodium ion/proton antiporter NhaA from Escherichia coli (see figures). The crystals of one other transporter diffract to high resolution, and a good native data set has been collected to a resolution of 2.8 Å. Structure determination is underway. In parallel, the precise function of the transporters which yielded well diffracting crystals will be identified, either by trying to complement functionally deficient bacterial strains (e.g. E. coli deletion mutants), or by incorporating the purified transporters into liposomes and identification of the substrate transported. Mechanistic studies will be started with the transporter reconstituted into proteoliposomes and into supported lipid bilayer membranes. Transporters that cannot be produced in E. coli, or not in sufficient amounts, are being produced using a cell-free coupled transcription/translation system.

We would like to know the structures and the mechanism of transport of representative transporters, and in more general terms (i) which of the four source organisms leads to the highest rate of success, (ii) whether there are transporter families which can be more easily expressed than others, and (iii) whether the in vitro cell-free coupled transcription/translation system constitutes a scientifically and economically viable alternative to bacterial expression systems.

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