Albert Bondt Tessa Sinnige Laurens Vehmeijer Introduction Experiments Structural studies Functional studies Conclusion Discussion Membrane proteins: mostly -helices Outer membranes proteins Gram(-) bacteria, mitochondria and chloroplasts: mostly - barrels OMPs: Outer Membrane Proteins Synthesized in cytoplasm Transported to periplasm by SecYEG Transported to -barrel assembly sites on OM OMP structure probably recognized by assembly complex Folded and inserted by conserved process involving a multiprotein machine Four lipoproteins: YfgL, YfiO, NlpB and SmpA Conserved barrel: YaeT in E.coli, Sam50 in mitochondria, Toc75 in chloroplasts YaeT Essential for viability Reported to bind C-terminal peptides of OMPs Large region in the intermembrane space contains POTRA domains. POlypeptide TRansport-Associated (POTRA) domains Implicated role assembling other beta-barrel proteins in mitochondria Implicated role as docking sites for proteins to be transported over membrane in chloroplasts What is the structure of periplasmic part of YaeT? Which POTRA domains are essential? How do they bind different peptide sequences? Complete periplasmic fragment: YaeT All five POTRA domains Crystallization unsuccessful Partial periplasmic fragment: YaeT Only first four POTRA domains Crystallization successful Fishhook-like shape Successive POTRA domains rotated in right-handed fashion Similar secondary structures despite low sequence similarity Order: 1 - 1 - 2 - 2 - 3 Three -strands -sheet 1 and 2 : edges 3 : center Two antiparallel -helices Only hydrophobic core and loop regions conserved between POTRA domains Implicates importance for structure YaeT : dimer in crystal Intertwined monomers Solvent-accessible part is buried H-bonds at edge of P3 and first residues of P5 stump Only major contact area monomers Formation -strand parallel to 2 of P3 causes dimerization Formation -stranded interface may be needed for successful crystallization Dimer not physiologically relevant YaeT elutes as a monomer from SEC N-terminus P5 needed for -interface in YaeT not available in wt-protein Dimerization shows possible interaction of other proteins with POTRA domains -augmentation: addition of -strands to -sheet through H-bonds Similar highly ordered contacts at interfaces all POTRA domains fishhook confirmation in monomer P5 crucial for interactions with lipoproteins OMP assembly complex functions as monomer Blue-Native PAGE Ni 2+ -affinity chromatography All POTRA domains required for proper function -bulge P3 involved in interaction with YfgL Evidence for -augmentation P3 loop might interact with Imp POTRA domains have fold Domains form a fishhook arrangement POTRA domains can interact by augmentation P3 and P5 crucial for interactions Fishhook conformation native? Extensive hydrophobic and polar inter-domain contacts Fishhook conformation native? Probably not! More extended conformation shown by NMR, SAXS and X-ray Mechanism of YaeT? Monomer or oligomer Interactions with lipoproteins Recognition of substrate