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Spiess (1995) has suggested that the membrane topology of eukaryotic proteins may be controlled by the translocase or the associated TRAM component of the endoplasmic reticulum which recognizes the charge distribution of the protein regions. In many cases, insertion of proteins into the membrane requires the Sec machinery. Most prokaryotic and eukaryotic membrane proteins have an asymmetric distribution of charged amino acid residues flanking the transmembrane segments of the protein ( von Heijne and Gavel, 1988 Hartmann et al., 1989). However, it remains unknown which structural changes occur during the membrane insertion process and whether the charged amino acid residues are transported through the lipid bilayer in an ionic state with a hydration shell or whether they are neutralized by protonation or deprotonation. This suggests that the Pf3 protein undergoes considerable structural changes upon the insertion process. In organic solvent, Pf3 coat protein exists as a trimer with a 90% α-helical conformation, whereas in planar lipid bilayers it can form voltage-gated channels, probably as tetrameric units ( Pawlak et al., 1994). Recently, it was demonstrated that the Pf3 protein adopts a conformation in lipid vesicles that is 75% α-helical and that in aqueous solution it shows a 40% α-helical structure ( Thiaudiere et al., 1993). Due to its small size and simple membrane topology, the Pf3 coat is a very attractive system for the study of the fundamental mechanisms of membrane insertion and also for determining the structural properties which are important for this process. When expressed from a plasmid in Escherichia coli, it is inserted into the inner membrane with an N outC in orientation. The 44-amino-acid, single membrane-spanning (type III) coat protein of the Pseudomonas aeruginosa phage Pf3 has been studied as a model protein for Sec-independent membrane translocation (Kuhn et al., 1990 Rohrer and Kuhn, 1990 Lee et al., 1992 Cao and Dalbey, 1994). In addition, we show in vivo that the electrochemical membrane potential is necessary for the translocation of both the wild-type and the mutant Pf3 coat proteins, suggesting that membrane insertion is driven by electrophoretic forces. In both systems, the orientation of the protein was completely reversed for the oppositely charged mutant coat protein (RD mutant). Membrane insertion was analyzed in vivo using the accessibility to externally added protease and in vitro by testing the insertion into inverted Escherichia coli membrane vesicles. To investigate how the orientation of this protein is achieved, the three flanking charged amino acid residues were altered. The N-terminal sequence immediately flanking the membrane anchor contains one negatively charged residue, whereas the C-terminal hydrophilic segment has two positively charged residues. The coat protein of Pseudomonas aeruginosa phage Pf3 is transiently inserted into the bacterial inner membrane with a single transmembrane anchor sequence in the N outC in orientation. ![]() All items shown 'in stock' are in stock, in the UK and ready for processing and dispatch with no extra charges, duties or import tax. We are a UK company based in Stevenage for over 43 years. Hand wash and air-dry the Microclear™ bag regularly. Using it will preserve the superior optical quality of Oakley's pure Plutonite® lenses. To help maintain the High Definition Optics® (HDO®) of Oakley lenses, your new eyewear comes with a specially-woven electrostatic Microclear™ bag for cleaning and storage. 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