VAAM-Jahrestagung 2012 18.–21. März in Tübingen

66CEP004Investigation on the subcellular localization of the GramicidinS synthetaseM. Hartmann* 1 , M. Berditsch 1 , S. Afonin 2 , C. Weber 3 , M. FotouhiArdakani 4 , D. Gerthsen 4 , A.S. Ulrich 3,21 KIT/Institute of Organic Chemistry and CFN, Karlsruhe, Germany2 KIT/Institute of Biological Interfaces (IBG-2), Karlsruhe, Germany3 KIT/ Institute of Organic Chemistry, Biochemistry, Karlsruhe, Germany4 KIT/Laboratory for Electron Microscopy, DFG Center for FunctionalNanostructures, Karlsruhe, GermanyNon-ribosomal peptide synthetases (NRPS) enable bacterial and fungalcells to produce a variety of important compounds, like antimicrobialpeptides, cytotoxic surfactants or siderophores, as an alternative way to theribosomal peptide biosynthesis. We investigate the NRPS for Gramicidin S(GS), a cyclic ß-stranded decapeptide, which shows pronouncedantimicrobial activity against Gram-positive bacteria, and is also activeagainst Gram-negative bacteria, viruses and fungi. Like all NRPS, the GSsynthetase consists of two subunits, GrsA (127 kDa) and GrsB (510 kDa),which are each composed of several domains (A=adenylation,PCP=peptidylcarrier, C=condensation, And TE=thioester).Despite extensive research on the modular structure of NRPS, littleattention has been paid on its subcellular localization in the producingcells. Here, we investigated the localization of GS synthetaseinAneurinibacillus migulanus, and Western blot analysis was used tocompare cytosolic and membrane fractions of GS-producing and nonproducingphenotypes. Immuno-gold electron microscopy was performedwith antibodies against the A-domain of GrsA. These combined resultsshow that GS synthetase is localized in the membrane fractions. Based onhydropathy analysis of the A-domain, we then examined its affinitytowards different phospholipids. These lipid-protein interaction studiesshowed an affinity of the GrsA A-domain especially to cardiolipin, whichis present inA. migulanusmembranes in high concentration. Our resultssuggest that it will be possible to optimize the reconstitution of NRPS onsolid support materials for the production of peptidesin vitro.[1] Hoyer, K. M., C. Mahlert, and M. A. Marahiel.2007. The iterative gramicidin s thioesterasecatalyzes peptide ligation and cyclization. Chem Biol14:13-22[2] Snider, C., S. Jayasinghe, K. Hristova, and S. H. White.2009 MPEx: a tool for exploringmembrane proteins. Protein Sci18:2624-8.[3] Berditsch, M., S. Afonin, and A. S. Ulrich.2007. The ability ofAneurinibacillusmigulanus(Bacillus brevis) to produce the antibiotic gramicidin S is correlated with phenotypevariation. Appl Environ Microbiol73:6620-8.CEP005Influence of flotillins on lipid raft dynamicsJ. Bach*, M. BramkampInstitute of Biochemistry, University of Cologne, Cologne, GermanyBiological membranes are characterized by a high diversity of lipids.Contrary to previous assumptions it could be shown that these lipids arenot homogeneously distributed in the membrane but form highlyspecialized domains, also termed lipid rafts. In these lipid rafts particularproteins are present and can routinely be isolated with these lipid rafts.One subset of these proteins are flotillins. Flotillins normally contain ahairpin loop that tethers the protein to the membrane, accordingly flotillinsexhibit a SPFH (stomatin-prohibitin-Flotillin-HflK/C)-domain and aflotillin domain. Furthermore flotillins and other SPFH-domain containingproteins build highly dynamic oligomeric structures. However, thefunction of flotillins is not yet fully understood but it is generally assumedthat they act as scaffolding proteins for lipid rafts. In the living cell it issupposed that highly specialized proteins and lipids are recruited byflotillins to microdomains and form functional complexes. The closesthomologue to human flotillin1 can be found in the model organismBacillus subtilis. In previous work we were able to identify severalinteracting proteins of the flotillin homologue, namely YuaG (FloT).Detergent resistant membranes (DRM) were isolated from a strainexpressing SNAP-YuaG. The DRMs were incubated with magnetic beadslinked to benzylguanine that covalently binds to the SNAP-tag. Severalproteins that are likely interaction partner of YuaG were co-eluted.Strikingly, no crosslinking of these proteins was required for co-elution.One of the identified proteins is the SPFH-domain containing proteinYqfA. However, several other proteins were co-eluted with YuaG. Herewe show how the identified protein complexes functionally depend on theformation of lipid microdomains.CEP006Analysis of the chlamydial translation elongation factor EF-TuS. De Benedetti*, A. Gaballah, B. HenrichfreiseInstitute for Medical Microbiology, Immunology and Parasitology(IMMIP), Pharmaceutical Microbiology Section, Bonn, Germanyvital role in Bacillus subtilis: it contributes to cell shape maintenance,apparently via interaction with the cytoskeleton protein MreB. In rodshapedbacteria the actin-ortholog MreB is thought to direct incorporationof cell wall material into the side wall. Surprisingly, chlamydiae harbor,despite their spherical shape and the absence of a cell wall, MreB and werecently proved in vitro activity for this protein.Here, we show that EF-Tu from Chlamydophila pneumoniae is functionalin vitro. The purified, strep-tagged protein polymerized in a concentration,pH and ion strength dependent fashion in light scattering andsedimentation assays. Additionally, using co-pelleting assays, wedemonstrated that (i) chlamydial EF-Tu interacts with MreB and (ii) thepolymerization of MreB is improved in the presence of EF-Tu.A deeper insight into the functions of EF-Tu and its role in chlamydial cellbiology on molecular level will provide valuable information for thedesign of new anti-chlamydial antibiotics.CEP007Investigation of TatA d oligomerization to a pore complexC. Gottselig* 1 , T. Walther 2 , S. Vollmer 2 , F. Stockmar 3 , G.U. Nienhaus 3 ,A.S. Ulrich 1,21 KIT, Institute of Biological Interfaces 2, Karlsruhe, Germany2 KIT, Institute of Organic Chemistry, Karlsruhe, Germany3 KIT, Institute of Applied Physics, Karlsruhe, GermanyThe “twin arginine translocase” (Tat) is a protein export machinery thattransports certain folded proteins across the bacterial plasma membrane.The cargo-proteins are targeted to the Tat pathway via an N-terminalsignal sequence containing a distinctive twin-arginine motif. The Tatsystem of Bacillus subtilis consists of two essential components, the TatAand TatC proteins, where the transmembrane protein TatA has beensuggested to form a protein-conducting channel by self-assembly, but littleis known about its oligomeric structure or the translocation mechanism.We have recently discovered a conserved pattern of charged amino acidsthat are able to form a network of consecutive salt-bridges, and on thisbasis we proposed a three-dimensional model of the pore-forming complexTatA d. Our hypothesis is that TatA d could self-assemble via intramolecularand intermolecular salt bridges into tetramers, which can subsequentlyoligomerize to a pore complex with variable diameter. To test and confirmthis model of pore formation, we have produced different di-cysteinemutants to replace the postulated salt-bridges by covalent bridges, whichshould allow us to distinguish intra- and intermolecular contacts. Furthercharge mutants TatA d have been produced to analyze their effect on theoligomerization behavior by SDS-PAGE and Blue-Native PAGE. Singlecysteineside chains have also been introduced into TatA d, to whichfluorophores or spin labels can be covalently bound for FluorescenceCorrelation Spectroscopy (FCS), Förster Resonance Energy Transfer(FRET) and Electron Spin Resonance (ESR) experiments. The selfassemblyof TatA d monomers into an oligomeric pore complex is beingstudied using FCS, and distances between TatA d proteins will be detectedby FRET and ESR.CEP008S-Layer proteins as platform for nanoscale sensor applicationsO. Riebe*, C. Berger, H. BahlUniversität Rostock, Biowissenschaften/Mikrobiologie, Rostock, GermanyIn many prokaryotes Surface Layer (S-layer) proteins are the outermostsurface of the cell. These self-assembling protein layers have variousexciting features. The monomeric proteins are clustered on the cell surfacein an entropy-driven process and form paracrystalline highly regularstructures. Depending on the organism different arrangements of theprotein subunits are possible. They are composed from one to six identicalsubunits resulting in oblique (p1, p2), square (p4) or hexagonal forms (p3or p6) of the protein lattice. We investigated proteins with different latticesymmetries for the application in nanostructured sensor chips. Due to thevery regular organisation with an ample supply of functional groups (e. g.-NH 2 or SH groups), this lattices should function as the basic buildingblock for a nanosensor. The functionalisation of this sensor is managed bycrosslinking of the functional groups to specific receptors for chemicalcompounds based on Aptamers and a combination with fluorescent dyes.Thus, the sensor could be used for the detection of drugs or otherchemicals in fresh- or process water. Here, we present first results on themultimerisation- and binding characteristics of heterologously expressedS-layer fragments as well as coating and coupling experiments for their usein a novel detection system.The bacterial translation elongation factor EF-Tu is well known to beinvolveld in prokaryotic protein biosynthesis. EF-Tu from Escherichia colihas been shown to polymerize in vitro and a recent study providedevidence that the protein serves besides its function in translation anotherBIOspektrum | Tagungsband 2012