TtOmp85, a β-Barrel Assembly Protein, Functions by Barrel Augmentation

Mallarino, Luisa Estrada; Fan, Enguo; Odermatt, Meike; Müller, Matthias; Lin, Meng-Ting; Liang, Jie; Heinzelmann, Martin; Fritsche, F; Apell, Hans-Jürgen; Welte, Wolfram

doi:10.1021/bi5011305

Cited by 26 publications

(32 citation statements)

References 42 publications

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“…This was achieved by computing the partition function after exhaustive enumeration of the configuration states in the reduced state space, following ref [32, 34]. The energy of each configuration was evaluated using the Msip potential function, which incorporates both single residue burial energy and pairwise inter-strand interaction energies (see Materials and Methods for details) [32, 34, 38]. …”

Section: Resultsmentioning

confidence: 99%

Engineering a Novel Porin OmpGF Via Strand Replacement from Computational Analysis of Sequence Motif

Lin

Zhang

Fahie

et al. 2017

Biochimica et Biophysica Acta (BBA) - Biomembranes

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β-barrel membrane proteins (βMPs) form barrel-shaped pores in the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts. Because of the robustness of their barrel structures, βMPs have great potential as nanosensors for single-molecule detection. However, natural βMPs currently employed have inflexible biophysical properties and are limited in their pore geometry, hindering their applications in sensing molecules of different sizes and properties. Computational engineering has the promise to generate βMPs with desired properties. Here we report a method for engineering novel βMPs based on the discovery of sequence motifs that predominantly interact with the cell membrane and appear in more than 75% of transmembrane strands. By replacing β1–β6 strands of the protein OmpF that lack these motifs with β1–β6 strands of OmpG enriched with these motifs and computational verification of increased stability of its transmembrane section, we engineered a novel βMP called OmpGF. OmpGF is predicted to form a monomer with a stable transmembrane region. Experimental validations showed that OmpGF could refold in vitro with a pre dominant β-sheet structure, as confirmed by circular dichroism. Evidence of OmpGF membrane insertion was provided by intrinsic tryptophan fluorescence spectroscopy, and its pore-forming property was determined by a dye-leakage assay. Furthermore, single-channel conductance measurements confirmed that OmpGF function as a monomer and exhibits increased conductance than OmpG and OmpF. These results demonstrated that a novel and functional βMP can be successfully engineered through strand replacement based on sequence motif analysis and stability calculation.

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Section: Resultsmentioning

confidence: 99%

Engineering a Novel Porin OmpGF Via Strand Replacement from Computational Analysis of Sequence Motif

Lin

Zhang

Fahie

et al. 2017

Biochimica et Biophysica Acta (BBA) - Biomembranes

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“…However, there does not appear to be a TamA homolog in D. radiodurans , indicating that DR_146T might act as a solo TamB. The presence of TamB in other bacteria lacking TamA has been reported, and the TAM system is suggested to have evolved from an original combination of TamB and BamA, which evolved into TamA in Proteobacteria by a later gene duplication event152938. Whether there was any interaction between TamB and BamA in D. radiodurans is not clear.…”

Section: Discussionmentioning

confidence: 99%

A tamB homolog is involved in maintenance of cell envelope integrity and stress resistance of Deinococcus radiodurans

Dai

et al. 2017

Sci Rep

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The translocation and assembly module (TAM) in bacteria consists of TamA and TamB that form a complex to control the transport and secretion of outer membrane proteins. Herein, we demonstrated that the DR_1462-DR_1461-DR_1460 gene loci on chromosome 1 of Deinococcus radiodurans, which lacks tamA homologs, is a tamB homolog (DR_146T) with two tamB motifs and a DUF490 motif. Mutation of DR_146T resulted in cell envelope peeling and a decrease in resistance to shear stress and osmotic pressure, as well as an increase in oxidative stress resistance, consistent with the phenotype of a surface layer (S-layer) protein SlpA (DR_2577) mutant, demonstrating the involvement of DR_146T in maintenance of cell envelope integrity. The 123 kDa SlpA was absent and only its fragments were present in the cell envelope of DR_146T mutant, suggesting that DR_146T might be involved in maintenance of the S-layer. A mutant lacking the DUF490 motif displayed only a slight alteration in phenotype compared with the wild type, suggesting DUF490 is less important than tamB motif for the function of DR_146T. These findings enhance our understanding of the properties of the multilayered envelope in extremophilic D. radiodurans, as well as the diversity and functions of TAMs in bacteria.

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“…What of the alternative scenario, wherein TamB interacts with BamA in these organisms? There have been functional analyses of BamA from organisms with a TamB–BamA operon ( Lenhart and Akins 2010 ; Lenhart et al 2012 ), or a TamB encoded on the genome separately from BamA, but again without TamA in the genome ( Nesper et al 2008 ; Tripp et al 2012 ; Estrada Mallarino et al 2015 ). Although these studies have not reported BamA–TamB interactions, neither have they excluded the formation of a TamB–BamA complex or more transient interaction between the proteins.…”

Section: Discussionmentioning

confidence: 99%

Evolution of the Translocation and Assembly Module (TAM)

Heinz

Selkrig

Belousoff

et al. 2015

Genome Biology and Evolution

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Bacterial outer membrane proteins require the beta-barrel assembly machinery (BAM) for their correct folding and function. The central component of this machinery is BamA, an Omp85 protein that is essential and found in all Gram-negative bacteria. An additional feature of the BAM is the translocation and assembly module (TAM), comprised TamA (an Omp85 family protein) and TamB. We report that TamA and a closely related protein TamL are confined almost exclusively to Proteobacteria and Bacteroidetes/Chlorobi respectively, whereas TamB is widely distributed across the majority of Gram-negative bacterial lineages. A comprehensive phylogenetic and secondary structure analysis of the TamB protein family revealed that TamB was present very early in the evolution of bacteria. Several sequence characteristics were discovered to define the TamB protein family: A signal-anchor linkage to the inner membrane, beta-helical structure, conserved domain architecture and a C-terminal region that mimics outer membrane protein beta-strands. Taken together, the structural and phylogenetic analyses suggest that the TAM likely evolved from an original combination of BamA and TamB, with a later gene duplication event of BamA, giving rise to an additional Omp85 sequence that evolved to be TamA in Proteobacteria and TamL in Bacteroidetes/Chlorobi.

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TtOmp85, a β-Barrel Assembly Protein, Functions by Barrel Augmentation

Cited by 26 publications

References 42 publications

Engineering a Novel Porin OmpGF Via Strand Replacement from Computational Analysis of Sequence Motif

Engineering a Novel Porin OmpGF Via Strand Replacement from Computational Analysis of Sequence Motif

A tamB homolog is involved in maintenance of cell envelope integrity and stress resistance of Deinococcus radiodurans

Evolution of the Translocation and Assembly Module (TAM)

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