Affinity-chromatographic purification of sixteen cysteine-substituted maltoporin variants: thiol reactivity and cross-linking in an outer membrane protein of Escherichia coli

Francis, G.; Brennan, L A; Ferenci, Thomas

doi:10.1016/0005-2736(91)90029-8

Cited by 16 publications

(9 citation statements)

References 27 publications

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“…The ability of the mutant strains to bind dextrins was then examined by affinity chromatography on starch–Sepharose columns, as described previously (Francis et al ., 1991a,b; Klebba et al ., 1994). The wild‐type strain AC1 and all the LamB mutants were grown in glucose minimal medium and induced with 4 × 10 −5 M IPTG to ensure sufficient expression of the LamB protein at the bacterial surface to allow binding to the columns.…”

Section: Resultsmentioning

confidence: 99%

In vivo and in vitro studies of major surface loop deletion mutants of the Escherichia coli K‐12 maltoporin: contribution to maltose and maltooligosaccharide transport and binding

Andersen

Bachmeyer

Täuber

et al. 1999

Molecular Microbiology

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SummaryThe trimeric protein LamB of Escherichia coli K-12 (maltoporin) speci®cally facilitates the diffusion of maltose and maltooligosaccharides through the outer membrane. Each monomer consists of an 18-stranded antiparallel b-barrel with nine surface loops (L1 to L9). The effects on transport and binding of the deletion of some of the surface loops or of combinations of several of them were studied in vivo and in vitro. In vivo, single-, DL4, DL5, DL6, and double-loop deletions, DL4 DL5 and DL5 DL6, abolished maltoporin functions, but not the double deletion DL4 DL6 and the triple deletion DL4 DL5 DL6. While deletion of the central variable portion of loop L9 (DL9v) affected maltoporin function only moderately, the combination of DL9v with the double deletion of loops L4 and L6 (triple deletion DL4 DL6 DL9v) strongly impaired maltoporin function and resulted in sensitivity to large hydrophilic antibiotics without change in channel size as measured in vitro. In vitro, the carbohydrate-binding properties of the different loop mutants were studied in titration experiments using the asymmetric and symmetric addition of the mutant porins and of the carbohydrates to one or both sides of the lipid bilayer membranes. The deletion of loop L9v alone (LamBDL9v), of two loops L4 and L6 (LamBDL4 DL6), of three loops L4, L5 and L6 (LamBDL4 DL5 DL6) or of L4, L6 and L9v (LamBDL4 DL6 DL9v) had relatively little in¯uence on the carbohydrate-binding properties of the mutant channels, and they had approximately similar binding properties for carbohydrate addition to both sides compared with only one side. The deletion of one of the loops L4 (LamBDL4) or L6 (LamBDL6) resulted in an asymmetric carbohydrate binding. The in vivo and in vitro results, together with those of the puri®cation across the starch column, suggest that maltooligosaccharides enter the LamB channel from the cell surface side with the non-reducing end in advance. The absence of some of the loops leads to obstruction of the channel from the outside, which results in a considerable difference in the on-rate of carbohydrate binding from the extracellular side compared with that from the periplasmic side.

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Section: Resultsmentioning

confidence: 99%

In vivo and in vitro studies of major surface loop deletion mutants of the Escherichia coli K‐12 maltoporin: contribution to maltose and maltooligosaccharide transport and binding

Andersen

Bachmeyer

Täuber

et al. 1999

Molecular Microbiology

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“…To further demonstrate the ability of the O-tRNA and mutant GlnRS to serve as a translationally active system in vivo, as well as establish a screen for the incorporation of a keto amino acid by an orthogonal aminoacyl-tRNA synthetase, two amber mutants of the gene encoding the E. coli maltoporin LamB were constructed by PCR mutagenesis. The sites of these amber mutations, positions Asn-72 and Asp-200, both lie within loops of LamB, which are known from an x-ray crystal structure (31) and from biochemical studies (32) to exist in the extracellular space. In addition, insertion of the C3 epitope from poliovirus at these sites results in the display of this epitope on the surface of the cell in a manner accessible to anti-C3 polyclonal and monoclonal antibodies (33).…”

Section: Fig 2 Flow Chart Describing the History Of Each Glnrs Librmentioning

confidence: 99%

Engineering a tRNA and aminoacyl-tRNA synthetase for the site-specific incorporation of unnatural amino acids into proteins in vivo

Liu

Magliery²,

Pastrnak³

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

175

121

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In an effort to expand the scope of protein mutagenesis, we have completed the first steps toward a general method to allow the site-specific incorporation of unnatural amino acids into proteins in vivo. Our approach involves the generation of an ''orthogonal'' suppressor tRNA that is uniquely acylated in Escherichia coli by an engineered aminoacyl-tRNA synthetase with the desired unnatural amino acid. To this end, eight mutations were introduced into tRNA 2Gln based on an analysis of the x-ray crystal structure of the glutaminyl-tRNA aminoacyl synthetase (GlnRS)-tRNA 2 Gln complex and on previous biochemical data. The resulting tRNA satisfies the minimal requirements for the delivery of an unnatural amino acid: it is not acylated by any endogenous E. coli aminoacyl-tRNA synthetase including GlnRS, and it functions efficiently in protein translation. Repeated rounds of DNA shuffling and oligonucleotidedirected mutagenesis followed by genetic selection resulted in mutant GlnRS enzymes that efficiently acylate the engineered tRNA with glutamine in vitro. The mutant GlnRS and engineered tRNA also constitute a functional synthetase-tRNA pair in vivo. The nature of the GlnRS mutations, which occur both at the protein-tRNA interface and at sites further away, is discussed.

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“…It has previously been demonstrated that a Ser-57--3Cys mutant had phenotypes no different from those of the wild type (12), which is also consistent with the tolerance of these residues in accepting neutral substitutions. However, labeling studies on the mutant carrying the Ser-57--*Cys mutation showed that the thiol was not as accessible as expected for a freely external loop (11). In OmpF, there is no membrane-external loop but a tight beta turn at the corresponding position.…”

Section: Resultsmentioning

confidence: 90%

Combinatorial mutagenesis of the lamB gene: residues 41 through 43, which are conserved in Escherichia coli outer membrane proteins, are informationally important in maltoporin structure and function

Chan

Ferenci

1993

J Bacteriol

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A new strategy for combinatorial mutagenesis was developed and applied to residues 40 through 60 of LamB protein (maltoporin), with the aim of identifying amino acids important for LamB structure and function. The strategy involved a template containing a stop codon in the target sequence and a pool of random degenerate oligonucleotides covering the region. In vitro mutagenesis followed by selection for function (Dex+, ability to utilize dextrins) corrected the nonsense mutation and simultaneously forced incorporation of a random mutation(s) within the region. The relative importance of each residue within the target was indicated by the frequency and nature of neutral and deleterious mutations recovered at each position. Residues 41 through 43 in LamB accepted few neutral substitutions, whereas residues 55 through 57 were highly flexible in this regard.Consistent with this finding was that the majority of defective mutants were altered at residues 41 to 43.Characterization of these mutants indicated that the nature of residues 41 to 43 influenced the amount of stable protein in the outer membrane. These results, as well as the conserved nature of this stretch of residues among outer membrane proteins, suggest that residues 41 to 43 of LamB play an important role in the process of outer membrane localization.Combinatorial mutagenesis studies are becoming important in the identification of important residues of proteins, including transport proteins (3,15,23,25,26). In this study, we developed a novel strategy for introducing random mutations into a defined region of maltoporin (LamB protein), with the aim of identifying residues significant in contributing to structure, assembly, and sugar transport. The strategy used with LamB can be applied to the mutagenesis of any gene coding for a selectable phenotype and has considerable advantages over cassette mutagenesis methods.Maltoporin in the outer membrane of Escherichia coli has been favored as a model in studies of phage (lambda) binding (4) and sugar channel selectivity (1) as well as protein export and assembly (9,20). Amino acid residues which play an important role in phage lambda and sugar binding have been identified (4,14). However, the events leading to the localization and assembly of LamB trimers into the outer membrane are still not clear. In addition to the signal sequence, specific regions in the mature protein may also play an important role in the biogenesis process (9, 20) but remain to be convincingly identified.There are several lines of evidence to suggest that residues 40 through 60 of mature LamB are of structural importance. Short in-frame deletions overlapping residues 39 to 49 of mature LamB resulted in the formation of unstable protein that was rapidly degraded (24), possibly as a result of incorrect outer membrane routing. Apart from its potential role in localization, the N-terminal third of the sequence has also been postulated to be important for sugar selectivity and channel formation (14,29) (Fig. 1) in outer membrane proteins such ...

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Affinity-chromatographic purification of sixteen cysteine-substituted maltoporin variants: thiol reactivity and cross-linking in an outer membrane protein of Escherichia coli

Cited by 16 publications

References 27 publications

In vivo and in vitro studies of major surface loop deletion mutants of the Escherichia coli K‐12 maltoporin: contribution to maltose and maltooligosaccharide transport and binding

In vivo and in vitro studies of major surface loop deletion mutants of the Escherichia coli K‐12 maltoporin: contribution to maltose and maltooligosaccharide transport and binding

Engineering a tRNA and aminoacyl-tRNA synthetase for the site-specific incorporation of unnatural amino acids into proteins in vivo

Combinatorial mutagenesis of the lamB gene: residues 41 through 43, which are conserved in Escherichia coli outer membrane proteins, are informationally important in maltoporin structure and function

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