Outer membrane protein

Because of the limited number of functions of the outer membrane, it consists of much fewer protein components than does the cytoplasmic membrane.As will be discussed later, the major outer membrane proteins exist in extremely large quantities, of the order of 1-8 x 10 molecules/cell, suggesting that they occupy a considerable part of the outer membrane structure. This unique feature of the outer membrane provides us with an excellent system for the investigation of the mechanism of biosynthesis and the mode of assembly of individual outer membrane proteins, their interaction with other proteins, phospholipids, and lipopolysaccharide, and their functions. 

The purpose of this article is not to review the numerous reports in this field, but to describe the approach for elucidating the problems alluded to above by focusing on the work undertaken in the author s laboratory. 

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Many small molecules can penetrate the outer ced membrane by diffusion through channels created by outer-membrane proteins caded porins. 

Despite considerable efforts very few membrane proteins have yielded crystals that diffract x-rays to high resolution. In fact, only about a dozen such proteins are currently known, among which are porins (which are outer membrane proteins from bacteria), the enzymes cytochrome c oxidase and prostaglandin synthase, and the light-harvesting complexes and photosynthetic reaction centers involved in photosynthesis. In contrast, many other membrane proteins have yielded small crystals that diffract poorly, or not at all, using conventional x-ray sources. However, using the most advanced synchrotron sources (see Chapter 18) it is now possible to determine x-ray structures from protein crystals as small as 20 pm wide which will permit more membrane protein structures to be elucidated. 

Gram-negative bacteria are surrounded by two membranes, an inner plasma membrane and an outer membrane. These are separated by a periplasmic space. Most plasma membrane proteins contain long, continuous sequences of about 20 hydrophobic residues that are typical of transmembrane a helices such as those found in bacteriorhodopsin. In contrast, most outer membrane proteins do not show such sequence patterns. 

This enigma was resolved in 1990 when the x-ray structure of an outer membrane protein, porin, showed that the transmembrane regions were p... 

Overexpression of apoaequorin (Inouye et al., 1989, 1991). To produce a large quantity of apoaequorin, an apoaequorin expression plasmid piP-HE containing the signal peptide coding sequence of the outer membrane protein A (ompA) of E. coli (Fig. 4.1.12) was constructed and expressed in E. coli. The expressed apoaequorin was secreted into the periplasmic space of bacterial cells and culture medium. The cleaving of ompA took place during secretion thus the... 

Furthermore, if the antibiotic passes membranes through a specific port of entry, its mutational loss leads to resistance. The lack of the outer membrane protein OprD in P. aeruginosa causes resistance to the (3-lactam antibiotic imipenem. Fosfomycin passes the cytoplasmic membrane via an L-a-glycerol phosphate permease. This transport system is not essential for bacterial growth and therefore mutants with a reduced expression are frequently selected under therapy. 

Kersten, G. F. A., Van de Put, A., Teerlink, T., Beuvery, E. C., and Crommelin, D. J. A. (1988a). Immunogenicity of liposomes and iscoms containing the major outer membrane protein of Neisseria gonorrhoeae -Influence of protein content and liposomal bilayer composition. Inf. Immun., 56. 1661-1664. 

The mechanism of acquired resistance in Pseudomonas aeruginosa is different. Chromosomal mutations result in the increase of a specific outer membrane protein with a concomitant reduction in divalent cations. Polymyxins bind to the outer membrane at sites normally occupied by divalent cations, and therefore it is thought that a reduction in these sites will lead to decreased binding of the antibiotic with a consequent decreased susceptibility of the cell. 

T. Ojanen, 1. M. Helander, K. Haahtela, T. K. Korhnonen, and T. Laak.so, Outer membrane proteins and lipopolysaccharides in pathovars of Xanthomonas camp-estris, Appl. Environ. Microbiol. 59 4143 (1993). 

Hansson, M., Samuelson, P., Nguyen, T.N. and Stahl, S. (2002) General expression vectors for Staphylococcus carnosus enabled efficient production of the outer membrane protein A of Klebsiella pneumoniae. FEMS Microbiology Letters, 210 (2), 263—270. 

Mainly the outer membrane ferrichrome receptor and transporter FhuA will be discussed because most structural and functional studies have been performed with this protein. In fact, FhuA was the first outer membrane protein identified (called TonA), with known functions as a phage and colicin receptor, that are related to iron transport (for a historical account, see Braun and Hantke 1977). 

Zhang H,Tang X, Munske GR, et al. In vivo identification of the outer membrane protein OmcA-MtrC interaction network in Shewanella oneidensis MR-1 cells using novel hydrophobic chemical cross-linkers. J. Proteome Res. 2008 7 1712-1720. 

Newhall, J., Sawyer, W.D., and Haak, R.A. (1980) Cross-linking analysis of the outer membrane proteins of Neisseria gonorrhoeae. Infect. Immun. 28, 785-791. 

PRP-OMP PedvaxHIB (Merck) Neisseria meningitidis serogroup B outer membrane protein... 

There are several hypotheses for a specific mechanism by which ONOO- can control the open state of the PTPC. Briefly the PTPC is regulated by primary constituents of the pore, including the inner membrane adenine nucleotide translocase (ANT) and the outer membrane protein voltage-dependent anion channel (VDAC or porin). The VDAC-ANT complex can bind to signaling proteins that modulate permeability transition, such as pro-apoptotic Bax (which opens the pore) and anti-apoptotic Bcl-2... 

Outer membrane proteins face several problems. They have to cross the inner membrane thus implying that they are not allowed to contain hydrophobic membrane anchor or stop transfer sequences. Moreover, correct targeting to and stable insertion into the outer membrane is important for proper functioning. A number of OM proteins assemble into oligomers even prior to insertion. 

Based on sequence similarity, it appears that transporters of this type may also assist in the uptake and acquisition of solutes unrelated to iron chelating compounds and vitamin Bi2 [68], Examples of prospective candidates are outer membrane proteins that are involved in sulfate ester utilisation in Pseudomonas putida [69], or polypeptides playing a role in starch binding at the surface of Bacteriodes thetaiotaomicron [70]. 

As mentioned above, transport of siderophores across the cytoplasmic membrane is less specific than the translocation through the outer membrane. In E. coli three different outer membrane proteins (among them FepA the receptor for enterobactin produced by most E. coli strains) recognise siderophores of the catechol type (enterobactin and structurally related compounds), while only one ABC system is needed for the passage into the cytosol. Likewise, OM receptors FhuA, FhuE, and Iut are needed to transport a number of different ferric hydroxamates, whereas the FhuBCD proteins accept a variety of hydroxamate type ligands such as albomycin, ferrichrome, coprogen, aerobactin, shizokinen, rhodotorulic acid, and ferrioxamine B [165,171], For the vast majority of systems, the substrate specificity has not been elucidated, but it can be assumed that many siderophore ABC permeases might be able to transport several different but structurally related substrates. 

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