Membrane topology and assembly of the outer membrane protein OmpA of Escherichia coli K12

Ried, Georg; Koebnik, Ralf; Hindennach, Ingrid; Mutschler, Bettina; Henning, Ulf

doi:10.1007/bf00280309

Cited by 70 publications

(50 citation statements)

References 45 publications

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“…A similar result was already described for OmpA digestion by pronase, where a unique 19-kDa protease-resistant fragment was found, corresponding to the N-terminal part [19]. The resistance of the N-terminal part of OmpA to protease treatments is likely to be due to a very tight structural domain, as suggested by the high proportion of antiparallel L-strands in this protein [20,21]. This hypothesis was recently con- ¢rmed by the crystal structure of the N-terminal fragment 1^171 of E. coli OmpA (OmpA 171 ) revealing that this domain consists unambiguously of an extended eightstranded L-barrel [14].…”

Section: Resultssupporting

confidence: 79%

Ion channel formation by N-terminal domain: a common feature of OprFs of Pseudomonas and OmpA of Escherichia coli

Saint¹

2000

FEMS Microbiology Letters

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The proteolytic fragments of OprFs of Pseudomonas aeruginosa and Pseudomonas fluorescens were identified, respectively, as the first 175 and 177 amino acids from the N-terminal domain. They induced ion channels after reincorporation into planar lipid bilayers (85 and 75 pS, respectively, in 1 M NaCl). A similar conductance value (72 pS) was found for the eight L-strand OmpA N-terminal domain (OmpA 171 ) of Escherichia coli. We conclude that the N-terminal domain of OprFs is sufficient to induce ion channels and the comparison with OmpA 171 , provides strong evidence of the existence of an eight-stranded L-barrel in the N-terminal domain of OprFs. ß

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Section: Resultssupporting

confidence: 79%

Ion channel formation by N-terminal domain: a common feature of OprFs of Pseudomonas and OmpA of Escherichia coli

Saint¹

2000

FEMS Microbiology Letters

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“…The protein termini are usually located in the periplasmic space, as found with the porins. Both turns and loops are often permissive for the insertion of small peptides (Charbit et al, 1991;Koebnik and Braun, 1993;Ried et al, 1994). Taken together, these models suggest some common structural and/or folding principles.…”

Section: Introductionmentioning

confidence: 89%

“…The process of membrane assembly of outer membrane proteins in vivo is only poorly understood (for a recent review, see Henning and Koebnik, 1994). The Escherichia coli outer membrane protein OmpA is well suited for the study of this problem (Freudl et al, 1986;Klose et al, 1988Klose et al, , 1993Ried et al, 1994;Koebnik and Kramer, 1995). Although its three-dimensional structure has not yet been solved, a detailed model has been proposed by means of phage mapping, linker insertion mutagenesis in combination with protease digestion experiments, Raman spectroscopy and computer-based predictions (Morona et al, 1984;Vogel and Jahnig, 1986;Ried et al, 1994).…”

Section: Introductionmentioning

confidence: 99%

“…The Escherichia coli outer membrane protein OmpA is well suited for the study of this problem (Freudl et al, 1986;Klose et al, 1988Klose et al, , 1993Ried et al, 1994;Koebnik and Kramer, 1995). Although its three-dimensional structure has not yet been solved, a detailed model has been proposed by means of phage mapping, linker insertion mutagenesis in combination with protease digestion experiments, Raman spectroscopy and computer-based predictions (Morona et al, 1984;Vogel and Jahnig, 1986;Ried et al, 1994). This model predicts a two-domain organization of OmpA with 170 N-terminal amino acids residing in the outer membrane and the remaining 155 C-terminal residues extending into the periplasmic space (Figure 1).…”

Section: Introductionmentioning

confidence: 99%

“…It is thought that the eight 5-strands form a ,8-barrel. Recently, a truncated variant consisting of only the 171 N-terminal amino acid residues has been constructed and shown to be assembled efficiently into the outer membrane in vivo (Ried et al, 1994). For the study of membrane assembly, this variant has the advantages over the porins of being a monomeric protein and of having a much smaller membrane moiety.…”

Section: Introductionmentioning

confidence: 99%

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In vivo membrane assembly of split variants of the E.coli outer membrane protein OmpA.

Koebnik

1996

The EMBO Journal

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The two‐domain, 325 residue outer membrane protein OmpA of Escherichia coli is a well‐established model for the study of membrane assembly. The N‐terminal domain, consisting of approximately 170 amino acid residues, is embedded in the membrane, presumably in the form of a beta‐barrel consisting of eight antiparallel transmembrane beta‐strands. A set of 16 gene variants carrying deletions in the membrane‐embedded domain of OmpA was constructed. When pairs of these mutant genes were co‐expressed in E.coli, it was found that a functional OmpA protein could be assembled efficiently from two complementary protein fragments. Assembly was found when the polypeptide chain was split at the second or third periplasmic turn. All four protein termini were located in the periplasmic space. Interestingly, duplication of transmembrane strands five and six led to a variant with an unusual topology: the N‐terminus of one fragment and the C‐terminus of the other fragment were exposed at the cell surface. This is the first demonstration of correct membrane assembly of split beta‐structured membrane proteins. These findings are important for a better understanding of their folding/assembly pathway and may have implications for the development of artificial outer membrane proteins and for the cell surface display of heterologous peptides or proteins.

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Strategy for membrane protein crystallization exemplified with OmpA and OmpX

et al. 1999

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The bacterial outer membrane proteins OmpA and OmpX were modified in such a manner that they yielded bulky crystals diffracting X-rays isotropically beyond 2 Å resolution and permitting detailed structural analyses. The procedure involved semi-directed mutagenesis, mass production into inclusion bodies, and (re)naturation therefrom; it should be applicable for a broader range of membrane proteins. Proteins 1999;34:167-172.1999 Wiley-Liss, Inc.

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Membrane topology and assembly of the outer membrane protein OmpA of Escherichia coli K12

Cited by 70 publications

References 45 publications

Ion channel formation by N-terminal domain: a common feature of OprFs of Pseudomonas and OmpA of Escherichia coli

Ion channel formation by N-terminal domain: a common feature of OprFs of Pseudomonas and OmpA of Escherichia coli

In vivo membrane assembly of split variants of the E.coli outer membrane protein OmpA.

Strategy for membrane protein crystallization exemplified with OmpA and OmpX

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