Genetic mapping of starch‐ and Lambda‐receptor sites in maltoporin: identification of substitutions causing direct and indirect effects on binding sites by cysteine mutagenesis

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

doi:10.1111/j.1365-2958.1991.tb02160.x

Cited by 26 publications

(24 citation statements)

References 22 publications

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“…1) (4), which suggested that these residues constitute a membrane-external loop. 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).…”

Section: Resultssupporting

confidence: 60%

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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Section: Resultssupporting

confidence: 60%

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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“…Neither the sucrose porin ScrY nor the nonspecific E. coli porins conserve these residues to the extent found in OprB, suggesting a possible role in binding maltodextrins; a characteristic common to both OprB and LamB. Residue W74 in LamB and conserved in OprB and ScrY as W71 and W151, respectively, has also been specifically linked to maltodextrin binding (Charbit et al, 1988;Francis et al, 1991), suggesting some of the conserved residues within this region are involved in the formation of a binding site. Further studies of these residues within OprB and LamB are warranted to compare and contrast the effects on the structure and function of each of these porins.…”

Section: Discussionmentioning

confidence: 99%

Cloning and nucleotide sequence of the Pseudomonas aeruginosa glucose‐selective OprB porin gene and distribution of OprB within the family Pseudomonadaceae

Wylie

Worobec

1994

European Journal of Biochemistry

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OprB is a glucose-selective porin known to be produced by Pseudomonas aeruginosa and Pseudomonas putida. We have cloned and sequenced the oprB gene of I? aeruginosa and obtained expression of OprB in Escherichia coli. The mature protein consists of 423 amino acid residues with a deduced molecular mass of 47597 Da. Several clusters of amino acid residues, potentially involved in the structure or function of the protein, were identified. An area of regional homology with E. coli LamB was also identified. Carbohydrate-inducible proteins, potentially homologous to OprB, were identified in several rRNA homology-group-I pseudomonads by sodium dodecyl sulfate/ polyacrylamide gel electrophoresis analysis, Western immunoblotting and N-terminal amino acid sequencing. These species also contained DNA that hybridized to a P. aeruginosa oprB gene probe.Porin proteins form trans-membrane water-filled channels in the outer membrane of Gram-negative bacteria and allow the diffusion of substrates between the external environment and the periplasm of the cell (Nikaido and Vaara, 1985). Two types of porins have been recognized; nonspecific porins and substrate-selective porins. Nonspecific porins form channels which allow nonspecific diffusion of substrates based on solute mass, charge and polarity. Substrateselective porins show some substrate specificity and possess a binding site for a given substrate within the channel. In comparison to nonspecific porins, very few substrate-selective porins have been identified and characterized.The glucose-selective porin, OprB, of Pseudomonas aeruginosa was first identified by Hancock and Carey (1980) following SDSPAGE analysis of outer membranes of cells grown on glucose as the sole carbon source. Porin function was demonstrated by the efflux of several carbohydrates from vesicles reconstituted from purified OprB, lipopolysaccharide and phospholipid. Trias et al. (1988), using the liposome-swelling assay, characterized OprB as a substrate-selective porin by demonstrating selectivity of OprB for glucose and xylose. Subsequently, a binding site for glucose and several other carbohydrates was demonstrated in P. aeruginosa OprB and its homologue in Pseudomonas putida by Wylie et al. (1993) and Saravolac et al. (1991), respectively. f? aeruginosa OprB bound glucose with a K, of 380 mM. K, for glucose binding to I? putida OprB was within the same order of magnitude, with a value of 110 mM. P. putida OprB was also shown to bind maltose and maltotriose with a higher affinity than glucose, although neither l? aeruginosa nor P. putida is able to utilize maltose or maltotriose as a carbon source. Single-channel-conductance measurements indicated that both P. aeruginosa and P. putida OprB form small constricted channels [25 pS (picosiemens) for P. aeruginosa within the pore of these proteins. Surprisingly, although I? aeruginosa and I? putida OprB appear closely related based on immunological and biochemical evidence (Saravolac et al., 1991 ; Wylie et al., 1993), the two porins show opposite ion sel...

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“…Enhanced uptake of starch breakdown products through the LamB channel is likely to give the enteric bacteria a competitive advantage. A large assortment of lamB mutants has been used to probe the roles of different parts of the protein [7, 8, 9]. Once the x-ray structure provided details of its β-barrel topology and the residues lining the “greasy slide” that confer specificity to the pore [10, 11, 12], genetic analysis of the architecture of LamB protein could be targeted to determining structure-function relationships of various loops and pore residues [13, 14].…”

Section: Introductionmentioning

confidence: 99%

Folding studies of purified LamB protein, the maltoporin from the Escherichia coli outer membrane: Trimer dissociation can be separated from unfolding

Baldwin

Bhatia

Luckey

2011

Biochimica et Biophysica Acta (BBA) - Biomembranes

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The folding mechanisms for β-barrel membrane proteins present unique challenges because acquisition of both secondary and tertiary structure is coupled with insertion into the bilayer. For the porins in Escherichia coli outer membrane, the assembly pathway also includes association into homotrimers. We study the folding pathway for purified LamB protein in detergent and observe extreme hysteresis in unfolding and refolding, as indicated by the shift in intrinsic fluorescence. The strong hysteresis is not seen in unfolding and refolding a mutant LamB protein lacking the disulfide bond, as it unfolds at much lower denaturant concentrations than wild type LamB protein. The disulfide bond is proposed to stabilize the structure of LamB protein by clasping together the two sides of Loop 1 as it lines the inner cavity of the barrel. In addition we find that low pH promotes dissociation of the LamB trimer to folded monomers, which run at about one third the size of the native trimer during SDS PAGE and are much more resistant to trypsin than the unfolded protein. We postulate the loss at low pH of two salt bridges between Loop 2 of the neighboring subunit and the inner wall of the monomer barrel destabilizes the quaternary structure.

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Genetic mapping of starch‐ and Lambda‐receptor sites in maltoporin: identification of substitutions causing direct and indirect effects on binding sites by cysteine mutagenesis

Cited by 26 publications

References 22 publications

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

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

Cloning and nucleotide sequence of the Pseudomonas aeruginosa glucose‐selective OprB porin gene and distribution of OprB within the family Pseudomonadaceae

Folding studies of purified LamB protein, the maltoporin from the Escherichia coli outer membrane: Trimer dissociation can be separated from unfolding

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