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.
Kohlstaedt M, von der Hocht I, Hilbers F, Thielmann Y, Michel H (2015) Development of a Thermofluor assay for stability determination of membrane proteins using the Na+/H+ antiporter NhaA and cytochrome c oxidase. Acta Cryst D 71, 1112-22.
Kaur J, Olkhova E, Nand Malviya V, Grell E, Michel H (2014) A L-Lysine Transporter of High Stereoselectivity of the APC Superfamily: Production, Functional Characterisation and Structure Modelling. J Biol Chem 289, 1377-1387.
Xie H, Buschmann S, Langer JD, Ludwig B, Michel H (2014) Biochemical and biophysical characterization of the two isoforms of cbb3-type cytochrome c oxidase from Pseudomonas stutzeri. J Bacteriol 196, 472-82.
Jaehme M, Michel H (2013) Evaluation of cell-free protein synthesis for the crystallization of membrane proteins--a case study on a member of the glutamate transporter family from Staphylothermus marinus. FEBS J 280, 1112-1125.
Rührer S, Michel H (2013) Exploiting Leishmania tarentolae cell-free extracts for the synthesis of human solute carriers. Mol Membr Biol 30, 288-302.
Goswami G, Kaur J, Surade S, Grell E, Michel H (2012) Heterologous production and functional and thermodynamic characterization of cation diffusion facilitator (CDF) transporters of mesophilic and hyperthermophilic origin. J Biol Chem 393, 617-629.
Rycovska A, Hatahet L, Fendler K, Michel H (2012) The nitrite transport protein NirC from Salmonella typhimurium is a nitrite/proton antiporter. Biochim Biophys Acta 1818, 1342-1350.
Hedderich T, Marcia M, Koepke J, Michel H (2011) PICKScreens, a new database for the comparison of crystallization screens for biological macromolecules. Cryst Growth Des 11, 488-491.
Buschmann S, Warkentin E, Xie H, Langer JD, Ermler U, Michel H (2010) The structure of cbb3 cytochrome oxidase provides insights into proton pumping. Science 329, 327-330.
Marcia M, Ermler U, Peng G, Michel H (2009) The structure of Aquifex aeolicus sulfide:quinone oxidoreductase, a basis to understand sulfide detoxification and respiration. Proc Natl Acad Sci USA 106, 9625-9630.