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.     

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.     

The Escherichia coli outer membrane protein OmpA acquires secondary structure prior to its integration into the membrane

Almost all proteins that reside in the outer membrane (OM) of Gram-negative bacteria contain a membrane-spanning segment that folds into a unique β barrel structure and inserts into the membrane by an unknown mechanism. To obtain further insight into outer membrane protein (OMP) biogenesis, we revisited the surprising observation reported over 20 years ago that the Escherichia coli OmpA β barrel can be assembled into a native structure in vivo when it is expressed as two noncovalently linked fragments. Here, we show that disulfide bonds between β strand 4 in the N-terminal fragment and β strand 5 in the C-terminal fragment can form in the periplasmic space and greatly increase the efficiency of assembly of “split” OmpA, but only if the cysteine residues are engineered in perfect register (i.e., they are aligned in the fully folded β barrel). In contrast, we observed only weak disulfide bonding between β strand 1 in the N-terminal fragment and β strand 8 in the C-terminal fragment that would form a closed or circularly permutated β barrel. Our results not only demonstrate that β barrels begin to fold into a β-sheet-like structure before they are integrated into the OM but also help to discriminate among the different models of OMP biogenesis that have been proposed.     

Liprotides assist in folding of outer membrane proteins

Proteins and lipids can form complexes called liprotides, in which the partially denatured protein forms a shell encasing a lipid core. This effectively stabilizes a lipid micelle in an aqueous solvent and suggests that liprotides may provide a suitable vessel for membrane proteins. Accordingly we have investigated if liprotides consisting of α‐lactalbumin and oleate could aid folding of four different outer membrane proteins (OMPs) tOmpA, PagP, BamA, and OmpF. tOmpA was able to fold in the presence of the liprotide, and folding did not occur if only oleate or α‐lactalbumin were added. Although the liprotides did not fold the other three OMPs on its own, it was able to assist their folding in the presence of vesicles. Incubation with liprotides before folding into vesicles increased the folding yield of the outer membrane proteins to a level higher than using micelles of the non‐ionic surfactant DDM. Even though the liprotide was stable at both high urea concentrations and high pH, it failed to efficiently fold OmpA at high pH. Instead, optimal folding was seen at pH 8–9, suggesting that important changes in the liprotide occurred when increasing the pH. We conclude that an otherwise folding‐inactive fatty acid can be activated when presented by a liprotide and thereby work as an in vitro chaperone for outer membrane proteins.     

Insights into outer membrane protein crystallization

Outer membrane proteins are structurally distinct from those that reside in the inner membrane and play important roles in bacterial pathogenicity and human metabolism. X-ray crystallography studies on >40 different outer membrane proteins have revealed that the transmembrane portion of these proteins can be constructed from either β-sheets or less commonly from α-helices. The most common architecture is the β-barrel, which can be formed from either a single anti-parallel sheet, fused at both ends to form a barrel or from multiple peptide chains. Outer membrane proteins exhibit considerable rigidity and stability, making their study through x-ray crystallography particularly tractable. As the number of structures of outer membrane proteins increases a more rational approach to their crystallization can be made. Herein we analyse the crystallization data from 53 outer membrane proteins and compare the results to those obtained for inner membrane proteins. A targeted sparse matrix screen for outer membrane protein crystallization is presented based on the present analysis.     

One membrane protein,two structures and six environments: a comparative molecular dynamics simulation study of the bacterial outer membrane protein PagP

PagP is a bacterial outer membrane protein consisting of an 8 stranded transmembrane β-barrel and an N-terminal α-helix. It is an enzyme which catalyses transfer of a palmitoyl chain from a phospholipid to lipid A. Molecular dynamics simulations have been used to compare the dynamic behaviour in simulations starting from two different structures (X-ray vs. NMR) and in six different environments (detergent micelles formed by dodecyl phosphocholine and by octyl glucoside, vs. four species of phospholipid bilayer). Analysis of interactions between the protein and its environment reveals the role played by the N-terminal α-helix, which interacts with the lipid headgroups to lock the PagP molecule into the bilayer. The PagP β-barrel adopts a tilted orientation in lipid bilayers, facilitating access of lipid tails into the mouth of the central binding pocket. In simulations starting from the X-ray structure in lipid bilayer, the L1 and L2 loops move towards one another, leading to the formation of a putative active site by residues H33, D76 and S77 coming closer together.     

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.     

Protein oligomerization in the bacterial outer membrane (Review)

The formation of homo-oligomeric assemblies is a well-established characteristic of many soluble proteins and enzymes. Oligomerization has been shown to increase protein stability, allow allosteric cooperativity, shape reaction compartments and provide multivalent interaction sites in soluble proteins. In comparison, our understanding of the prevalence and reasons behind protein oligomerization in membrane proteins is relatively sparse. Recent progress in structural biology of bacterial outer membrane proteins has suggested that oligomerization may be as common and versatile as in soluble proteins. Here we review the current understanding of oligomerization in the bacterial outer membrane from a structural and functional point of view.     

NMR structural studies of the bacterial outer membrane protein OmpX in oriented lipid bilayer membranes

The β-barrels found in the outer membranes of prokaryotic and eukaryotic organisms constitute an important functional class of proteins. Here we present solid-state NMR spectra of the bacterial outer membrane protein OmpX in oriented lipid bilayer membranes. We show that OmpX is folded in both glass-supported oriented lipid bilayers and in lipid bicelles that can be magnetically oriented with the membrane plane parallel or perpendicular to the direction of the magnetic field. The presence of resolved peaks in these spectra demonstrates that OmpX undergoes rotational diffusion around an axis perpendicular to the membrane surface. A tightly hydrogen-bonded domain of OmpX resists exchange with D₂O for days and is assigned to the transmembrane β-barrel, while peaks at isotropic resonance frequencies that disappear rapidly in D₂O are assigned to the extracellular and periplasmic loops. The two-dimensional ¹H/¹⁵N separated local field spectra of OmpX have several resolved peaks, and agree well with the spectra calculated from the crystal structure of OmpX rotated with the barrel axis nearly parallel (5° tilt) to the direction of the magnetic field. The data indicate that it will be possible to obtain site-specific resonance assignments and to determine the structure, tilt, and rotation of OmpX in membranes using the solid-state NMR methods that are currently being applied to α-helical membrane proteins.     

UGO1 encodes an outer membrane protein required for mitochondrial fusion

Membrane fusion plays an important role in controlling the shape, number, and distribution of mitochondria. In the yeast Saccharomyces cerevisiae, the outer membrane protein Fzo1p has been shown to mediate mitochondrial fusion. Using a novel genetic screen, we have isolated new mutants defective in the fusion of their mitochondria. One of these mutants, ugo1, shows several similarities to fzo1 mutants. ugo1 cells contain numerous mitochondrial fragments instead of the few long, tubular organelles seen in wild-type cells. ugo1 mutants lose mitochondrial DNA (mtDNA). In zygotes formed by mating two ugo1 cells, mitochondria do not fuse and mix their matrix contents. Fragmentation of mitochondria and loss of mtDNA in ugo1 mutants are rescued by disrupting DNM1, a gene required for mitochondrial division. We find that UGO1 encodes a 58-kD protein located in the mitochondrial outer membrane. Ugo1p appears to contain a single transmembrane segment, with its NH(2) terminus facing the cytosol and its COOH terminus in the intermembrane space. Our results suggest that Ugo1p is a new outer membrane component of the mitochondrial fusion machinery.     

Characterization of the outer membrane protein OprF of Pseudomonas aeruginosa in a lipopolysaccharide membrane by computer simulation

The N-terminal domain of outer membrane protein OprF of Pseudomonas aeruginosa forms a membrane spanning eight-stranded antiparallel beta-barrel domain that folds into a membrane channel with low conductance. The structure of this protein has been modeled after the crystal structure of the homologous protein OmpA of Escherichia coli. A number of molecular dynamics simulations have been carried out for the homology modeled structure of OprF in an explicit molecular model for the rough lipopolysaccharide (LPS) outer membrane of P. aeruginosa. The structural stability of the outer membrane model as a result of the strong electrostatic interactions compared with simple lipid bilayers is restricting both the conformational flexibility and the lateral diffusion of the porin in the membrane. Constricting side-chain interactions within the pore are similar to those found in reported simulations of the protein in a solvated lipid bilayer membrane. Because of the strong interactions between the loop regions of OprF and functional groups in the saccharide core of the LPS, the entrance to the channel from the extracellular space is widened compared with the lipid bilayer simulations in which the loops are extruding in the solvent. The specific electrostatic signature of the LPS membrane, which results in a net intrinsic dipole across the membrane, is found to be altered by the presence of OprF, resulting in a small electrically positive patch at the position of the channel.     

Modeling and simulations of a bacterial outer membrane protein: OprF from Pseudomonas aeruginosa

OprF is a major outer membrane protein from Pseudomonas aeruginosa, a homolog of OmpA from Escherichia coli. The N-terminal domains of both proteins have been demonstrated to form low conductance channels in lipid bilayers. Homology models, consisting of an eight-stranded beta-barrel, of the N-terminal domain OprF have been constructed based on the crystal structure of the corresponding domain from E. coli OmpA. OprF homology models have been evaluated via a set (6 x 10 ns) of simulations of the beta-barrel embedded within a solvated dimyristoyl-phosphatidylcholine (DMPC) bilayer. The conformational stability of the models is similar to that of the crystal structure of OmpA in comparable simulations. There is a degree of water penetration into the pore-like center of the OprF barrel. The presence of an acidic/basic (E8/K121) side-chain interaction within the OprF barrel may form a gate able to close/open a central pore. Lipid-protein interactions within the simulations were analyzed and revealed that aromatic side-chains (Trp, Tyr) of OprF interact with lipid headgroups. Overall, the behavior of the OprF model in simulations supports the suggestion that this molecule is comparable to OmpA. The simulations help to explain the mechanism of formation of low conductance pores within the outer membrane.     

Controlling the folding efficiency of an integral membrane protein

Research into the folding mechanisms of integral membrane proteins lags far behind that of water-soluble proteins, to the extent that the term protein folding is synonymous with water-soluble proteins. Hydrophobic membrane proteins, and particularly those with transmembrane alpha-helical motifs, are frequently considered too difficult to work with. We show that the stored curvature elastic stress of lipid bilayers can be used to guide the design of efficient folding systems for these integral membrane proteins. The curvature elastic stress of synthetic phosphatidylcholine/phosphatidylethanolamine lipid bilayers can be used to control both the rate of folding and the yield of folded protein. The use of a physical bilayer property generalises this approach beyond the particular chemistry of the lipids involved.     

Discrimination of outer membrane proteins using machine learning algorithms

Discriminating outer membrane proteins (OMPs) from other folding types of globular and membrane proteins is an important task both for identifying OMPs from genomic sequences and for the successful prediction of their secondary and tertiary structures. In this work, we have analyzed the performance of different methods, based on Bayes rules, logistic functions, neural networks, support vector machines, decision trees, etc. for discriminating OMPs. We found that most of the machine learning techniques discriminate OMPs with similar accuracy. The neural network-based method could discriminate the OMPs from other proteins [globular/transmembrane helical (TMH)] at the fivefold cross-validation accuracy of 91.0% in a dataset of 1,088 proteins. The accuracy of discriminating globular proteins is 88.8% and that of TMH proteins is 93.7%. Further, the neural network method is tested with globular proteins belonging to 30 different folding types and it could successfully exclude 95% of the considered proteins. The proteins with SAM domain such as knottins, rubredoxin, and thioredoxin folds are eliminated with 100% accuracy. These accuracy levels are comparable to or better than other methods in the literature. We suggest that this method could be effectively used to discriminate OMPs and for detecting OMPs in genomic sequences.     

Surprisingly high correlation between early and late stages in non-two-state protein folding

We collected quantitative kinetic data on early and late stages of folding in non-two-state proteins from the literature, and studied the relationship between the kinetics of the two stages. There was a surprisingly high correlation between the rate constants of these stages. The correlation coefficient of the logarithmic rate constants was as high as 0.97, which could not be caused by chance. We also studied relationships of the logarithmic rate constants of the two stages with native three-dimensional structures represented by the residue-residue contact map. There were again surprisingly high correlations between the logarithmic rate constants and the number of non-local contact clusters obtained from the contact maps. Because the number of non-local contact clusters represents overall arrangement of substructures in a native protein, the results strongly suggested the importance of the arrangement of the substructures for the kinetics of both early and late stages of protein folding.     

Gibbs motif sampling: detection of bacterial outer membrane protein repeats.

The detection and alignment of locally conserved regions (motifs) in multiple sequences can provide insight into protein structure, function, and evolution. A new Gibbs sampling algorithm is described that detects motif-encoding regions in sequences and optimally partitions them into distinct motif models; this is illustrated using a set of immunoglobulin fold proteins. When applied to sequences sharing a single motif, the sampler can be used to classify motif regions into related submodels, as is illustrated using helix-turn-helix DNA-binding proteins. Other statistically based procedures are described for searching a database for sequences matching motifs found by the sampler. When applied to a set of 32 very distantly related bacterial integral outer membrane proteins, the sampler revealed that they share a subtle, repetitive motif. Although BLAST (Altschul SF et al., 1990, J Mol Biol 215:403-410) fails to detect significant pairwise similarity between any of the sequences, the repeats present in these outer membrane proteins, taken as a whole, are highly significant (based on a generally applicable statistical test for motifs described here). Analysis of bacterial porins with known trimeric beta-barrel structure and related proteins reveals a similar repetitive motif corresponding to alternating membrane-spanning beta-strands. These beta-strands occur on the membrane interface (as opposed to the trimeric interface) of the beta-barrel. The broad conservation and structural location of these repeats suggests that they play important functional roles.