Categorization of Escherichia coli outer membrane proteins by dependence on accessory proteins of the β-barrel assembly machinery complex
2023; Elsevier BV; Volume: 299; Issue: 7 Linguagem: Inglês
10.1016/j.jbc.2023.104821
ISSN1083-351X
AutoresNakajohn Thewasano, Edward M. Germany, Yuki Maruno, Yukari Nakajima, Takuya Shiota,
Tópico(s)Enzyme Structure and Function
ResumoThe outer membrane (OM) of gram-negative bacteria is populated by various outer membrane proteins (OMPs) that fold into a unique β-barrel transmembrane domain. Most OMPs are assembled into the OM by the β-barrel assembly machinery (BAM) complex. In Escherichia coli, the BAM complex is composed of two essential proteins (BamA and BamD) and three nonessential accessory proteins (BamB, BamC, and BamE). The currently proposed molecular mechanisms of the BAM complex involve only essential subunits, with the function of the accessory proteins remaining largely unknown. Here, we compared the accessory protein requirements for the assembly of seven different OMPs, 8- to 22-stranded, by our in vitro reconstitution assay using an E. coli mid-density membrane. BamE was responsible for the full efficiency of the assembly of all tested OMPs, as it enhanced the stability of essential subunit binding. BamB increased the assembly efficiency of more than 16-stranded OMPs, whereas BamC was not required for the assembly of any tested OMPs. Our categorization of the requirements of BAM complex accessory proteins in the assembly of substrate OMPs enables us to identify potential targets for the development of new antibiotics. The outer membrane (OM) of gram-negative bacteria is populated by various outer membrane proteins (OMPs) that fold into a unique β-barrel transmembrane domain. Most OMPs are assembled into the OM by the β-barrel assembly machinery (BAM) complex. In Escherichia coli, the BAM complex is composed of two essential proteins (BamA and BamD) and three nonessential accessory proteins (BamB, BamC, and BamE). The currently proposed molecular mechanisms of the BAM complex involve only essential subunits, with the function of the accessory proteins remaining largely unknown. Here, we compared the accessory protein requirements for the assembly of seven different OMPs, 8- to 22-stranded, by our in vitro reconstitution assay using an E. coli mid-density membrane. BamE was responsible for the full efficiency of the assembly of all tested OMPs, as it enhanced the stability of essential subunit binding. BamB increased the assembly efficiency of more than 16-stranded OMPs, whereas BamC was not required for the assembly of any tested OMPs. Our categorization of the requirements of BAM complex accessory proteins in the assembly of substrate OMPs enables us to identify potential targets for the development of new antibiotics. Gram-negative bacteria contain an outer membrane (OM), a permeable barrier, and an inner (cytoplasmic) membrane. Outer membrane proteins (OMPs), a major component of the OM, are responsible for maintaining integrity and performing various functions in the OM (1Klebba P.E. Newton S.M. Mechanisms of solute transport through outer membrane porins: burning down the house.Curr. Opin. Microbiol. 1998; 1: 238-247Crossref PubMed Google Scholar, 2Nikaido H. Nakae T. The outer membrane of Gram-negative bacteria.Adv. Microb. Physiol. 1979; 20: 163-250Crossref PubMed Scopus (300) Google Scholar). A common characteristic of OMPs is the transmembrane domain, a β-barrel that folds into a single large cylinder of antiparallel β-sheets (3Misra R. Assembly of the β-barrel outer membrane proteins in Gram-negative bacteria, mitochondria, and chloroplasts.ISRN Mol. Biol. 2012; 2012708203Crossref PubMed Google Scholar). In Gram-negative bacteria, the β-barrel domains of all OMPs are composed of an even number of β-strands, providing a thermodynamically stable antiparallel network for the β-barrel. OMPs can be broadly classified into two groups (i) monopolypeptide barrels (e.g., OmpC) (4Baslé A. Rummel G. Storici P. Rosenbusch J.P. Schirmer T. Crystal structure of osmoporin OmpC from E. coli at 2.0 A.J. Mol. Biol. 2006; 362: 933-942Crossref PubMed Scopus (168) Google Scholar) or (ii) multipolypeptide barrels (e.g., GspD) (5Yan Z. Yin M. Xu D. Zhu Y. Li X. Structural insights into the secretin translocation channel in the type II secretion system.Nat. Struct. Mol. Biol. 2017; 24: 177-183Crossref PubMed Scopus (82) Google Scholar). Currently, monopolypeptide OMPs vary from 8- to 36-stranded, with species able to homo-oligomerize into monomers, dimers, or trimers (6Pautsch A. Schulz G.E. Structure of the outer membrane protein A transmembrane domain.Nat. Struct. Biol. 1998; 5: 1013-1017Crossref PubMed Scopus (419) Google Scholar, 7Vandeputte-Rutten L. Kramer R.A. Kroon J. Dekker N. Egmond M.R. Gros P. Crystal structure of the outer membrane protease OmpT from Escherichia coli suggests a novel catalytic site.EMBO J. 2001; 20: 5033-5039Crossref PubMed Scopus (214) Google Scholar, 8Barnard T.J. Gumbart J. Peterson J.H. Noinaj N. 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Bacterial outer membrane proteins are targeted to the bam complex by two parallel mechanisms.mBio. 2021; 12e00597-21Crossref Scopus (9) Google Scholar). Most monopolypeptide OMPs are inserted into the OM by the multisubunit molecular machine, known as the β-barrel assembly machinery (BAM) (16Ranava D. Caumont-Sarcos A. Albenne C. Ieva R. Bacterial machineries for the assembly of membrane-embedded β-barrel proteins.FEMS Microbiol. Lett. 2018; https://doi.org/10.1093/femsle/fny087Crossref PubMed Scopus (21) Google Scholar). The central component of the BAM complex is BamA, a member of the Omp85 protein superfamily, which is an essential protein conserved across bacterial lineages and eukaryotic organelles (17Wu T. Malinverni J. Ruiz N. Kim S. Silhavy T.J. Kahne D. Identification of a multicomponent complex required for outer membrane biogenesis in Escherichia coli.Cell. 2005; 121: 235-245Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar, 18Webb C.T. Heinz E. Lithgow T. Evolution of the β-barrel assembly machinery.Trends Microbiol. 2012; 20: 612-620Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Another essential subunit, BamD, is conserved across all gram-negative bacteria (19Anwari K. Webb C.T. Poggio S. Perry A.J. Belousoff M. Celik N. et al.The evolution of new lipoprotein subunits of the bacterial outer membrane BAM complex.Mol. Microbiol. 2012; 84: 832-844Crossref PubMed Scopus (46) Google Scholar). Furthermore, BamB, BamC, BamE, and BamF have been identified as subunits of the BAM complex (19Anwari K. Webb C.T. Poggio S. Perry A.J. Belousoff M. Celik N. et al.The evolution of new lipoprotein subunits of the bacterial outer membrane BAM complex.Mol. Microbiol. 2012; 84: 832-844Crossref PubMed Scopus (46) Google Scholar). They are necessary for efficient BAM complex function as "accessory subunits" but are not essential for bacterial viability. The composition of accessory subunits of the BAM complex varies in different subclasses of Gram-negative bacteria. For instance, the β-proteobacteria Neisseria meningitidis does not have BamB homologs (20Volokhina E.B. Beckers F. Tommassen J. Bos M.P. The beta-barrel outer membrane protein assembly complex of Neisseria meningitidis.J. Bacteriol. 2009; 191: 7074-7085Crossref PubMed Scopus (72) Google Scholar). In Escherichia coli (E. coli), the BAM complex is a heteropentameric complex composed of BamA, BamB, BamC, BamD, and BamE. The E. coli BamA, an OMP, contains five repeat polypeptide transport-associated (POTRA) domains upstream of the β-barrel transmembrane domain (21Kim S. Malinverni J.C. Sliz P. Silhavy T.J. Harrison S.C. Kahne D. Structure and function of an essential component of the outer membrane protein assembly machine.Science. 2007; 317: 961-964Crossref PubMed Scopus (295) Google Scholar). The POTRA domains are exposed in the periplasm and interact with four lipoproteins. Recent studies utilizing structural approaches have revealed the molecular mechanisms of the E. coli BAM complex from multiple atomic resolution structures (22Gu Y. Li H. Dong H. Zeng Y. Zhang Z. Paterson N.G. et al.Structural basis of outer membrane protein insertion by the BAM complex.Nature. 2016; 531: 64-69Crossref PubMed Google Scholar, 23Bakelar J. Buchanan S.K. Noinaj N. The structure of the β-barrel assembly machinery complex.Science. 2016; 351: 180-186Crossref PubMed Scopus (161) Google Scholar, 24Iadanza M.G. Higgins A.J. Schiffrin B. Calabrese A.N. Brockwell D.J. Ashcroft A.E. et al.Lateral opening in the intact β-barrel assembly machinery captured by cryo-EM.Nat. Commun. 2016; 712865Crossref PubMed Scopus (124) Google Scholar, 25Tomasek D. Rawson S. Lee J. Wzorek J.S. Harrison S.C. 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The formation of an antiparallel β-hairpin between the N-terminal strand of the lateral gate and C-terminal strand of the substrate proteins initiates the insertion step. Some structural studies have captured assembly intermediates, in which BamA and substrate proteins form a fully membrane-inserted hybrid barrel interlinked binding at the lateral gate. Prior to membrane insertion, the interior wall moiety of the BamD funnel interacts with the substrate protein and promotes partial β-sheet folding (29Hagan C.L. Wzorek J.S. Kahne D. Inhibition of the β-barrel assembly machine by a peptide that binds BamD.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 2011-2016Crossref PubMed Google Scholar). At present, all roles by the subunits BamA and BamD are deemed essential systems required for all OMPs, regardless of the character of the protein. In contrast, the roles of accessory proteins have not been assigned in this system. Several studies have revealed the function of these accessory proteins. BamB stabilizes the assembly precinct formed by the localization of multiple BAM complexes, enabling rapid oligomerization of homotrimeric OMPs such as OmpC by assembling multiple molecules in close proximity (30Gunasinghe S.D. Shiota T. Stubenrauch C.J. Schulze K.E. Webb C.T. Fulcher A.J. et al.The WD40 protein BamB mediates coupling of BAM complexes into assembly precincts in the bacterial outer membrane.Cell Rep. 2018; 23: 2782-2794Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). BamE stimulates conformational changes in the BAM complex (31Chen X. Ding Y. Bamert R.S. Le Brun A.P. Duff A.P. Wu C.-M. et al.Substrate-dependent arrangements of the subunits of the BAM complex determined by neutron reflectometry.Biochim. Biophys. Acta Biomembr. 2021; 1863183587Crossref Scopus (3) Google Scholar), and BamB and BamE double deletion E. coli strains became synthetically lethal (32Rigel N.W. Schwalm J. Ricci D.P. Silhavy T.J. BamE modulates the Escherichia coli beta-barrel assembly machine component BamA.J. Bacteriol. 2012; 194: 1002-1008Crossref PubMed Scopus (61) Google Scholar), indicating that both of these functions are important for assembly. However, a comprehensive understanding of how accessory proteins are involved in the assembly of various OMPs is lacking. In this study, we employed an in vitro reconstitution assay using an isolated mid-density membrane fraction (EMM; E. coli mid-density membrane). EMM is the membrane fraction of sonicated E. coli collected by high-speed centrifugation among differential centrifuges (low-speed, high-speed, and ultra-high-speed). We isolated EMMs from the accessory protein deletion and wildtype (WT) strains. To categorize the substrates according to the requirement of accessory proteins, we selected seven different monopolypeptide barrel-forming substrates, containing 8- to 22-stranded monomers, homodimers (two-pores), or homotrimers (three-pores). BamB was necessary for efficient assembly of OMPs with 16 or more β-strands and homotrimeric substrates. While the lack of BamC did not impair substrate assembly efficiently, BamE assisted in the proper assembly of all substrates, regardless of size or oligomeric state. Analysis of the state of the BAM complex showed that the lack of BamE destabilized the BAM complex formation, whereas BamB and BamC had limited effects. As all protein assemblies were impaired by BamE deletion, it is implied that BamE is important for the stable connection of BamD to BamA. Our analysis demonstrates the role of accessory proteins in the BAM complex, which can facilitate the assembly of a wide variety of substrates. These findings clarify which subunits should be targeted for the development of assembly inhibitors of pathogenic OMPs. Gram-negative bacteria have envelope stress response systems that maintain OM integrity. Since the deletion of the accessory proteins of the BAM complex induces the σE stress response, which regulates gene expression of the OMPs (periplasmic or chaperones) (33Rowley G. Spector M. Kormanec J. Roberts M. Pushing the envelope: extracytoplasmic stress responses in bacterial pathogens.Nat. Rev. Microbiol. 2006; 4: 383-394Crossref PubMed Scopus (252) Google Scholar), it is difficult for in vivo analysis to directly assess the functions of the accessory subunits. To overcome this problem, we employed an in vitro reconstitution assay using EMM containing intact BAM complexes. In this study, we deleted accessory proteins by gene disruption using a kanamycin cassette in BL21 (DE3)∗ as the parental strain. We isolated EMMs from each strain and then normalized them to the total protein level by the UV method. A comparison of the protein levels of subunits of the BAM complex at EMM equal total protein levels showed that deletion of accessory protein genes lost only itself but did not significantly affect the level of the other subunit proteins of the BAM complex (Fig. 1B). BamA levels in each EMM were not statistically different (Fig. S1), and we normalized the total protein level to assess the assembly efficiency. Adjusting the protein level of EMM between WT and mutant strains enabled us to compare the function of the BAM complex with minimal secondary effects, such as gene expression. EMM is composed of a native membrane formed by lipopolysaccharides and phospholipids, as well as an intact BAM complex. Thus, using EMMs isolated from each accessory protein deletion strain enabled us to assess the function of the BAM complex, which loses each accessory protein. Moreover, the levels of the major porins, OmpA, OmpC, and OmpF, in each EMM observed by the Coomassie brilliant blue (CBB) stain (34Prilipov A. Phale P.S. Van Gelder P. Rosenbusch J.P. Koebnik R. Coupling site-directed mutagenesis with high-level expression: large scale production of mutant porins from E. coli.FEMS Microbiol. Lett. 1998; 163: 65-72Crossref PubMed Google Scholar), corresponded to previous studies showing that ΔbamB or ΔbamE decreased the steady-state of major porins in the membrane fractions (Fig. 1C) (32Rigel N.W. Schwalm J. Ricci D.P. Silhavy T.J. BamE modulates the Escherichia coli beta-barrel assembly machine component BamA.J. Bacteriol. 2012; 194: 1002-1008Crossref PubMed Scopus (61) Google Scholar). For substrate proteins, we synthesized radiolabeled protein using methionine- or cysteine-containing radioactive isotopes, sulphur-35 (35S), by rabbit reticulocyte lysate. To assess only the assembly reaction performed by the BAM complex, all substrate proteins for the EMM assembly assay lacked the N-terminal SEC signal sequence (14Rapoport T.A. Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes.Nature. 2007; 450: 663-669Crossref PubMed Scopus (706) Google Scholar). Previously, we established the reconstitution of assembly into EMM of five different OMPs: major porin (OmpA) (6Pautsch A. Schulz G.E. Structure of the outer membrane protein A transmembrane domain.Nat. Struct. Biol. 1998; 5: 1013-1017Crossref PubMed Scopus (419) Google Scholar), autotransporter (EspP) (8Barnard T.J. Gumbart J. Peterson J.H. Noinaj N. Easley N.C. Dautin N. et al.Molecular basis for the activation of a catalytic asparagine residue in a self-cleaving bacterial autotransporter.J. Mol. Biol. 2012; 415: 128-142Crossref PubMed Scopus (35) Google Scholar), major porins (OmpC and OmpF) (9Phale P.S. Philippsen A. Kiefhaber T. Koebnik R. Phale V.P. Schirmer T. et al.Stability of trimeric OmpF porin: the contributions of the latching loop L2.Biochemistry. 1998; 37: 15663-15670Crossref PubMed Scopus (93) Google Scholar), and maltoporin (LamB) (10Dutzler R. Wang Y.F. Rizkallah P. Rosenbusch J.P. Schirmer T. Crystal structures of various maltooligosaccharides bound to maltoporin reveal a specific sugar translocation pathway.Structure. 1996; 4: 127-134Abstract Full Text Full Text PDF PubMed Google Scholar, 35Germany E.M. Ding Y. Imai K. Bamert R.S. Dunstan R.A. Nakajima Y. et al.Discovery of a conserved rule behind the assembly of β-barrel membrane proteins.bioRxiv. 2021; ([preprint])https://doi.org/10.1101/2021.10.29.466387Crossref Scopus (0) Google Scholar). In this study, we additionally established the assembly of two more OMPs: the integral OM protease (OmpT) (7Vandeputte-Rutten L. Kramer R.A. Kroon J. Dekker N. Egmond M.R. Gros P. Crystal structure of the outer membrane protease OmpT from Escherichia coli suggests a novel catalytic site.EMBO J. 2001; 20: 5033-5039Crossref PubMed Scopus (214) Google Scholar) and iron uptake system (CirA) (11Buchanan S.K. Lukacik P. Grizot S. Ghirlando R. Ali M.M.U. Barnard T.J. et al.Structure of colicin I receptor bound to the R-domain of colicin Ia: implications for protein import.EMBO J. 2007; 26: 2594-2604Crossref PubMed Scopus (82) Google Scholar). This allows for a comparison of the assembly efficiency for a diverse range and characteristics of OMPs (Table 1).Table 1Characteristics range of outer membrane proteins (OMPs) used in this studyProtein nameStructure (side)Structure (top)Number of β-strandOligomeric statePDB ID referenceOmpA8Monomer (Dimer)1BXW (6Pautsch A. Schulz G.E. Structure of the outer membrane protein A transmembrane domain.Nat. Struct. Biol. 1998; 5: 1013-1017Crossref PubMed Scopus (419) Google Scholar)OmpT10Monomer1I78 (7Vandeputte-Rutten L. Kramer R.A. Kroon J. Dekker N. Egmond M.R. Gros P. Crystal structure of the outer membrane protease OmpT from Escherichia coli suggests a novel catalytic site.EMBO J. 2001; 20: 5033-5039Crossref PubMed Scopus (214) Google Scholar)EspP12Monomer3SLJ (8Barnard T.J. Gumbart J. Peterson J.H. Noinaj N. Easley N.C. Dautin N. et al.Molecular basis for the activation of a catalytic asparagine residue in a self-cleaving bacterial autotransporter.J. Mol. Biol. 2012; 415: 128-142Crossref PubMed Scopus (35) Google Scholar)OmpC16Trimer2J4UOmpF16Trimer3O0E (9Phale P.S. Philippsen A. Kiefhaber T. Koebnik R. Phale V.P. Schirmer T. et al.Stability of trimeric OmpF porin: the contributions of the latching loop L2.Biochemistry. 1998; 37: 15663-15670Crossref PubMed Scopus (93) Google Scholar)LamB18Trimer1AF6 (10Dutzler R. Wang Y.F. Rizkallah P. Rosenbusch J.P. Schirmer T. Crystal structures of various maltooligosaccharides bound to maltoporin reveal a specific sugar translocation pathway.Structure. 1996; 4: 127-134Abstract Full Text Full Text PDF PubMed Google Scholar)CirA22Monomer2HDF (11Buchanan S.K. Lukacik P. Grizot S. Ghirlando R. Ali M.M.U. Barnard T.J. et al.Structure of colicin I receptor bound to the R-domain of colicin Ia: implications for protein import.EMBO J. 2007; 26: 2594-2604Crossref PubMed Scopus (82) Google Scholar)The 3D structure of the OMPs used in this study in side view and top view were provided with the characteristics of each OMPs in two categories, number of β-strands, and oligomeric state. This was followed by PDB ID access code. Open table in a new tab The 3D structure of the OMPs used in this study in side view and top view were provided with the characteristics of each OMPs in two categories, number of β-strands, and oligomeric state. This was followed by PDB ID access code. We analyzed the requirement of accessory proteins for OmpA, which has the fewest β-stranded OMPs (8-stranded OMPs) (Fig. 2A). As OmpA has a periplasmic exposed domain at the C terminus, the last strand of the barrel domain of OmpA is not located at the C terminus of proteins like typical OMPs. OmpA forms a dimer in the OM under specific conditions (36Whitelegge J. Gas-phase structure of the E. coli OmpA dimer.Structure. 2014; 22: 666-667Abstract Full Text Full Text PDF PubMed Google Scholar). OmpA was assigned as a monomeric OMP because OmpA dimerization requires a C-terminal domain. The insertion of the OmpA fold into the EMM via the BAM complex has been previously validated by a competition assay using the peptide of the range of the final strand of the OmpC, termed peptide 23, which inhibits the substrate recognition of the lateral gate of BamA (29Hagan C.L. Wzorek J.S. Kahne D. Inhibition of the β-barrel assembly machine by a peptide that binds BamD.Proc. Natl. Acad. Sci. U. S. 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Densitometry and comparison of folded OmpA showed that the assembly efficiency of OmpA in the EMM isolated from the BamE deletion strain was reduced to approximately 60% compared with other EMMs (Fig. 2A). OmpT is a 10-stranded monomeric OMP; while OmpT was used as a substrate protein for the reconstitution assay using purified BAM complex-embedded proteoliposomes (41Hagan C.L. Kim S. Kahne D. Reconstitution of outer membrane protein assembly from purified components.Science. 2010; 328: 890-892Crossref PubMed Scopus (212) Google Scholar), this study is the first to use OmpT for the EMM assembly assay. We validated whether the folding of OmpT into EMM occurred via the BAM complex following peptide 23 inhibition (Fig. 2B). Faster migration of OmpT significantly decreased in the presence of peptide 23, indicating that OmpT was assembled into the EMM via the BAM complex. We then compared the requirements of accessory proteins (Fig. 2C). The EMM isolated from the ΔbamE strain decreased the folding efficiency of OmpT to approximately 60%, as OmpA did. EspP, an autotransporter, contains two domains: the N-terminal passenger domain and C-terminal 12-stranded β-barrel domain. The passenger domain traverses the OM via a transiently formed asymmetric barrel of BamA with EspP during the assembly of the barrel domain, and then, the passenger domain is cleaved off by self-cleavage (42Ieva R. Tian P. Peterson J.H. Bernstein H.D. Sequential and spatially restricted interactions of assembly factors with an autotransporter domain.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: E383-E391Crossref PubMed Scopus (116) Google Scholar). This reaction can be reproduced with a short passenger domain, with most of it removed (43Leyton D.L. Johnson M.D. Thapa R. Huysmans G.H.M. Dunstan R.A. Celik N. et al.A mortise–tenon joint in the transmembrane domain modulates autotransporter assembly into bacterial outer membranes.Nat. Commun. 2014; 5: 4239Crossref PubMed Scopus (34) Google Scholar, 44Roman-Hernandez G. Peterson J.H. Bernstein H.D. Reconstitution of bacterial autotransporter assembly using purified components.Elife. 2014; 3e04234Crossref PubMed Scopus (74) Google Scholar). In this study, similar to our previous reports, we used a passenger domain with only 76 amino acids remaining close to the barrel domain, termed the precursor form. Because the barrel domain of EspP, which is properly inserted into EMM and removed the passenger domain, the mature form, became proteinase K (PK) resistant, we analyzed insertion efficiency by the amount of the PK-resistant self-cleaved form of EspP. The insertion efficiency of EspP into ΔbamE also decreased to approximately 60% compared to that of the others (Fig. 2D). These results suggest that among the accessory proteins, only BamE is needed for the efficient insertion of 8- to 12-stranded monomeric OMPs into the OM. OmpC and OmpF are 16-stranded β-barrel porins with a molecular weight of approximately 37 kDa (per monomer) and form stable homotrimers in the OM. As this homotrimer is resistant to SDS, OmpC, OmpF, or LamB can be observed at approximately 70 or 160 kDa by SDS-PAGE for heat modifiability, compared to the faster migration observed in monomeric OMPs (45Ureta A.R. Endres R.G. Wingreen N.S. Silhavy T.J. Kinetic analysis of the assembly of the outer membrane protein LamB in Escherichia coli mutants each lacking a secretion or targeting factor in a different cellular compartment.J. Bacteriol. 2007; 189: 446-454Crossref PubMed Scopus (72) Google Scholar, 46Hussain S. Peterson J.H. Bernstein H.D. Reconstitution of Bam complex-mediated assembly of a trimeric porin into proteoliposomes.mBio. 2021; 12e0169621Crossref Scopus (0) Google Scholar). Alternative methods to visualize trimeric OmpC or OmpF at a clearer resolution are through n-Dodecyl-β-D-maltoside (DDM) solubilization and analysis by
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