Topology of PhoE porin: the ‘eyelet’ region

Struyvé, M; Visser, J. W. M.; Adriaanse, Henriëtte; Benz, Roland; Tommassen, Jan

doi:10.1111/j.1365-2958.1993.tb01104.x

Cited by 49 publications

(34 citation statements)

References 46 publications

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“…Insertion of the 16-amino-acid epitope from Semliki Forest virus only strongly affected protein synthesis and folding at four of the 14 sites tested, thus adding it to the list of insertions (Charbit et al, 1986;Agterberg et al, 1990;LeClerc et al, 1991;Struyvé et al, 1993;Newton et al, 1996;Janssen and Tommassen, 1996) whose antigenic properties are fairly independent of the neighbouring amino acids and which only disturb natural protein folding a little.…”

Section: Discussionmentioning

confidence: 99%

Two‐dimensional structure of the Opc invasin from Neisseria meningitidis

Merker

Tommassen

Kušećek

et al. 1997

Molecular Microbiology

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SummaryA two-dimensional structural model was devised for the Opc outer membrane protein invasin which contains 10 transmembrane strands and five surfaceexposed loops. One continuous epitope recognized by three monoclonal antibodies was localized to the tip of loop 2 by synthetic peptides and site-directed mutagenesis while a second, discontinuous epitope recognized by a fourth antibody was localized to loops 4 and 5 by insertion mutagenesis. These monoclonal antibodies are bactericidal and inhibit adhesion and invasion. Most of the T-cell epitopes defined by Wiertz et al. (1996) were localized to the transmembrane strands. Oligonucleotides encoding a foreign epitope (ٌ) from Semliki Forest virus were inserted into Bgl II restriction sites created by site-directed mutagenesis. The ٌ epitopes inserted in all five predicted loops were recognized on the cell surface of live Escherichia coli bacteria by a monoclonal antibody and are exposed while ٌ epitopes in the N-terminus or three predicted turns were not. The results thus confirm important predictions of the model and define five permissive sites within surface-exposed loops which can be used to insert foreign epitopes.

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Section: Discussionmentioning

confidence: 99%

Two‐dimensional structure of the Opc invasin from Neisseria meningitidis

Merker

Tommassen

Kušećek

et al. 1997

Molecular Microbiology

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“…If this loop in OprP also folds back into the channel, an epitope inserted into it might not be accessible to the antibody and therefore might not be recognized as being at a surface-exposed region. The surface exposure of an epitope from the foot-and-mouth disease virus inserted into the third loop of PhoE was not achieved until three copies of the epitope were inserted (37), suggesting that this loop is somewhat shielded from the exterior surface of the cell.…”

Section: Discussionmentioning

confidence: 99%

“…The analogous loops in the Rhodobacter capsulatus porin and PhoE, OmpF, and OmpC have been shown to fold back into the channels and constrict their internal diameters (12,37,40). If this loop in OprP also folds back into the channel, an epitope inserted into it might not be accessible to the antibody and therefore might not be recognized as being at a surface-exposed region.…”

Section: Discussionmentioning

confidence: 99%

“…It is possible that the epitopes inserted at those sites were not efficiently exposed at the cell surface. Struyé et al similarly speculated that the reason that an epitope inserted into the second postulated loop of PhoE was not shown to be surface exposed (1) was because it was at the interface between a loop and an adjacent ␤-strand region (37).…”

Section: Discussionmentioning

confidence: 99%

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Insertion mutagenesis of the Pseudomonas aeruginosa phosphate-specific porin OprP

Sukhan

Hancock

1995

J Bacteriol

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The gene encoding the Pseudomonas aeruginosa phosphate-specific porin OprP was subjected to both linker and epitope insertion mutageneses. Nine of the 13 linker mutant genes expressed protein at levels comparable to those obtained with the wild-type gene. These mutant proteins were shown, by indirect immunofluorescence with an OprP-specific antiserum, to be properly exposed at the cell surface. Four of the linker mutant genes expressed protein at reduced levels which were not detectable at the cell surface. A foreign epitope from the circumsporozoite form of the malarial parasite Plasmodium falciparum was cloned into the linker sites of 12 of the 13 mutant genes. Seven of the resultant epitope insertion mutant genes expressed surface-exposed protein.Two of these mutant genes presented the foreign epitope at surface-accessible regions as assessed by indirect immunofluorescence with a malarial epitope-specific monoclonal antibody. The data from these experiments were used to create a topological model of the OprP monomer.The transport of hydrophilic molecules across the outer membranes of gram-negative bacteria is facilitated by a class of proteins known as porins. These proteins form water-filled, membrane-spanning channels which permit the passage of molecules through the membrane and into the periplasmic space (13). Porins have been divided into two categories, nonspecific or general diffusion porins and specific porins (29,30). General diffusion porins allow the passage of any hydrophilic molecule through their channels provided it is smaller than the channels' exclusion limit. Specific porins possess a substratebinding site which makes it possible, at rate-limiting concentrations, for them to transport molecules that bind to that site at a much higher rate than other molecules of comparable size. The structures of a number of general diffusion porins have been determined by both crystallographic (12, 40) and mutagenic (5, 10) techniques. They are composed of three identical subunits, each of which is a 16-stranded ␤-barrel. The ␤-strands are tilted at an angle with respect to the plane of the membrane and are connected by loops exposed to either the outer surface or the periplasmic space. The periplasmically exposed loops tend to be short and regular, while the surface-exposed loops are longer and more variable. The third surface loop actually folds back into the channel so as to constrict its internal diameter (12).While there exists a great deal of data regarding the structure of general porins, there is considerably less information pertaining to the structure of specific porins. The list of specific porins is rather short in comparison with that of general diffusion porins. They include LamB (26) and Tsx (20) in Escherichia coli and OprB (15), OprD (39), OprO (16), and OprP (17) in Pseudomonas aeruginosa. The maltoporin LamB is a well-studied example of a specific porin. This protein, like the general diffusion porins, is composed of three identical subunits, each of which has been proposed to be in the ...

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“…1). This residue is within the eyelet-forming loop, L3 (Cowan et al, 1992;Nikaido, 1992;Struyve et al, 1993) which folds into the barrel leading to the inaccessibility of the epitope on the cell surface. Loop L3 contains one and a half turns of a-helix, contributes to the constriction of the pore and is responsible for ion selectivity (Cowan e t al., 1992).…”

Section: Mab Reactivity With Cnbr-generated Fragmentsmentioning

confidence: 99%

Immunochemical structure of the OmpD porin from Salmonella typhimurium

Singh¹,

Miller²,

Williams³

et al. 1996

Microbiology

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The OmpD porin was isolated and purified from Salmonella typhimurium strain SH 7454 (ompC::TnlO), digested with cyanogen bromide (CNBr) and the peptide fragments were separated by SDS-PAGE. N-terminal sequencing identified a total of 96 residues from four distinct peptides. The sequence showed that OmpD is homologous to NmpC (75% identity), Lc (75%) and OmpC (70 YO) from Bcherichia coli, and OmpC (68%) from S. fjvphimurium . The sequence was essentially identical to the translated sequence of an nmpC-like gene of 5. typhimurium, currently placed at 3 8 6 centisomes of the chromosome. Our results and other data suggest, however, that this gene is actually the ompD gene, which is more correctly placed in the 34 centisome region of the chromosome. The CNBr-generated peptides were also screened with 16 anti-S. typhimurium OmpD monoclonal antibodies by Western blotting. These results, in conjunction with the prediction of the OmpD folding pattern based on the known three-dimensional structure of E. coli OmpF, showed a close immunological relationship among 5. fyphimurium OmpD and E. coli NmpC and Lc, and a strong conservation of sequences within the transmembrane Ip strands of these porins and E. coli OmpC, PhoE and OmpF, and Salmonella typhi OmpC.

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Topology of PhoE porin: the ‘eyelet’ region

Cited by 49 publications

References 46 publications

Two‐dimensional structure of the Opc invasin from Neisseria meningitidis

Two‐dimensional structure of the Opc invasin from Neisseria meningitidis

Insertion mutagenesis of the Pseudomonas aeruginosa phosphate-specific porin OprP

Immunochemical structure of the OmpD porin from Salmonella typhimurium

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