BamA forms a translocation channel for polypeptide export across the bacterial outer membrane

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The major outer membrane protein of Fusobacterium nucleatum (FomA) folds and inserts into lipid bilayers via parallel folding pathways

Membrane protein insertion and folding was studied for the major outer membrane protein of Fusobacterium nucleatum (FomA), which is a voltage-dependent general diffusion porin. The transmembrane domain of FomA forms a beta-barrel that is predicted to consist of 14 beta-strands. Here, unfolded FomA is shown to insert and fold spontaneously and quantitatively into phospholipid bilayers upon dilution of the denaturant urea, which was shown previously only for outer membrane protein A (OmpA) of Escherichia coli. Folding of FomA is demonstrated by circular dichroism and fluorescence spectroscopy, by SDS-polyacrylamide gel electrophoresis, and by single-channel recordings. Refolded FomA had a single-channel conductance of 1.1 nS at 1 M KCl, in agreement with the conductance of FomA isolated from membranes in native form. In contrast to OmpA, which forms a smaller eight-stranded beta-barrel domain, folding kinetics of the larger FomA were slower and provided evidence for parallel folding pathways of FomA into lipid bilayers. Two pathways were observed independent of membrane thickness with two different lipid bilayers, which were either composed of dicapryl phosphatidylcholine or dioleoyl phosphatidylcholine. This is the first observation of parallel membrane insertion and folding pathways of a beta-barrel membrane protein from an unfolded state in urea into lipid bilayers. The kinetics of both folding pathways depended on the chain length of the lipid and on temperature with estimated activation energies of 19 kJ/mol (dicapryl phosphatidylcholine) and 70 kJ/mol (dioleoyl phosphatidylcholine) for the faster pathways.     

POTRA Domains,Extracellular Lid,and Membrane Composition Modulate the Conformational Stability of the β Barrel Assembly Factor BamA

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Secondary and tertiary structure formation of the beta-barrel membrane protein OmpA is synchronized and depends on membrane thickness

The mechanism of membrane insertion and folding of a beta-barrel membrane protein has been studied using the outer membrane protein A (OmpA) as an example. OmpA forms an eight-stranded beta-barrel that functions as a structural protein and perhaps as an ion channel in the outer membrane of Escherichia coli. OmpA folds spontaneously from a urea-denatured state into lipid bilayers of small unilamellar vesicles. We have used fluorescence spectroscopy, circular dichroism spectroscopy, and gel electrophoresis to investigate basic mechanistic principles of structure formation in OmpA. Folding kinetics followed a second-order rate law and is strongly depended on the hydrophobic thickness of the lipid bilayer. When OmpA was refolded into model membranes of dilaurylphosphatidylcholine, fluorescence kinetics were characterized by a rate constant that was about fivefold higher than the rate constants of formation of secondary and tertiary structure, which were determined by circular dichroism spectroscopy and gel electrophoresis, respectively. The formation of beta-sheet secondary structure and closure of the beta-barrel of OmpA were correlated with the same rate constant and coupled to the insertion of the protein into the lipid bilayer. OmpA, and presumably other beta-barrel membrane proteins therefore do not follow a mechanism according to the two-stage model that has been proposed for the folding of alpha-helical bundle membrane proteins. These different folding mechanisms are likely a consequence of the very different intramolecular hydrogen bonding and hydrophobicity patterns in these two classes of membrane proteins.     

The bacterial outer membrane β-barrel assembly machinery

β-Barrel proteins found in the outer membrane of Gram-negative bacteria serve a variety of cellular functions. Proper folding and assembly of these proteins are essential for the viability of bacteria and can also play an important role in virulence. The β-barrel assembly machinery (BAM) complex, which is responsible for the proper assembly of β-barrels into the outer membrane of Gram-negative bacteria, has been the focus of many recent studies. This review summarizes the significant progress that has been made toward understanding the structure and function of the bacterial BAM complex.     

A combined kinetic push and thermodynamic pull as driving forces for outer membrane protein sorting and folding in bacteria

In vitro folding studies of outer membrane beta-barrels have been invaluable in revealing the lipid effects on folding rates and efficiencies as well as folding free energies. Here, the biophysical results are summarized, and these kinetic and thermodynamic findings are considered in terms of the requirements for folding in the context of the cellular environment. Because the periplasm lacks an external energy source the only driving forces for sorting and folding available within this compartment are binding or folding free energies and their associated rates. These values define functions for periplasmic chaperones and suggest a biophysical mechanism for the BAM complex.     

Crystal structure of Escherichia coli BamB, a lipoprotein component of the β-barrel assembly machinery complex

In Gram-negative bacteria, the BAM (β-barrel assembly machinery) complex catalyzes the essential process of assembling outer membrane proteins. The BAM complex in Escherichia coli consists of five proteins: one β-barrel membrane protein, BamA, and four lipoproteins, BamB, BamC, BamD, and BamE. Despite their role in outer membrane protein biogenesis, there is currently a lack of functional and structural information on the lipoprotein components of the BAM complex. Here, we report the first crystal structure of BamB, the largest and most functionally characterized lipoprotein component of the BAM complex. The crystal structure shows that BamB has an eight-bladed β-propeller structure, with four β-strands making up each blade. Mapping onto the structure the residues previously shown to be important for BamA interaction reveals that these residues, despite being far apart in the amino acid sequence, are localized to form a continuous solvent-exposed surface on one side of the β-propeller. Found on the same side of the β-propeller is a cluster of residues conserved among BamB homologs. Interestingly, our structural comparison study suggests that other proteins with a BamB-like fold often participate in protein or ligand binding, and that the binding interface on these proteins is located on the surface that is topologically equivalent to where the conserved residues and the residues that are important for BamA interaction are found on BamB. Our structural and bioinformatic analyses, together with previous biochemical data, provide clues to where the BamA and possibly a substrate interaction interface may be located on BamB.     

Indications of a common folding pattern for VDAC channels from all sources

Previous research on the mitochondrial channel VDAC from the yeastS. cerevisiae had identified protein strands forming the wall of VDAC's aqueous pore. Here we report the results of analyzing the primary sequences of VDAC from various sources to see if the transmembrane folding pattern identified from this yeast is conserved for VDAC of different species. We analyzed the primary sequences of VDAC from higher plants, fungi, invertebrates, and vertebrates and found that all have a very similar -partern profile with 12–15 peaks indicating potential sided beta strands that are candidates for protein strands forming the wall of the aqueous pore. All these VDAC sequences can be put into the 13 transmembrane strand folding pattern previously identified for yeast VDAC. These folding patterns agree with available experimental data: both electrophysiological and protease digestion data. Although the primary sequences of VDAC from very diverse organisms show low homology, sequence similarity in the proposed corresponding 13 transmembrane strands is substantial. Competing proposals utilizing 16 transmembrane strands are in conflict with electrophysiological experimental observations and violate the constraints on such strands, such as no charged amino acids facing the phospholipid membrane and sufficient number of residues to span the membrane.     

Isolation and characterization of the outer membrane of Acinetobacter calcoaceticus

A method for the separation of the outer membrane (OM) from the cytoplasmic membrane (CM) of Acinetobacter calcoaceticus 69/V grown on different carbon sources is described. The contamination of the OM with CM was less than 10%. Independent of the carbon source, five protein bands with apparent molecular weights of 47 000, 33000, 21 000, 19 000 and 12 000 were found by solubilization at 37°C and six bands at 100°C (apparent M_r 53 000, 47 000, 38 000, 26 000, 21000, 12000). Three proteins were modifiable by heat. With the periodic acid-Schiff procedure the bands with apparent M_r of 33 000 and 12 000 were made visible. After growth on d,l-carnitine an additional two non-heat-modifiable protein bands with apparent M_r between 40 000 and 45 000 were detected. By cultivation on acetate and peptone as carbon source one additional band (M_r 15 000) from OM of cells could be found.