Folding Of OmpA Research Articles

Novel Kinetic Intermediates Populated along the Folding Pathway of the Transmembrane β-Barrel OmpA.

We examined the folding of the β-barrel membrane protein OmpA from Escherichia coli. Although previous studies identified several intermediate states followed by a concerted translocation mechanism across the bilayer, some aspects of the pathway were still unclear, including the extent of secondary structure formation in the intermediate states and how the mechanism gave rise to multiple exponential phases in the folding kinetics. We addressed these questions by investigating the folding kinetics of the OmpA transmembrane β-barrel domain over a range of bilayer thicknesses, allowing us to observe different regions of the folding pathway. The fastest folding into the thinnest bilayers provided information about the later stages of the process, and the slowest folding into thicker bilayers revealed early kinetic steps. Folding was monitored using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and circular dichroism spectroscopy, which provide complementary information about tertiary and secondary structure formation. We globally fit the folding data to kinetic schemes and found that the same core pathway was followed under all lipid conditions. We propose a multistep folding mechanism for OmpA that includes unstructured surface-adsorbed states converting through a partially inserted state with substantial β-sheet structure to the final natively inserted barrel. Kinetic models show that all steps of the main folding pathway are accelerated by membrane defects that occur as a result of thinning the bilayer or incubation of lipids at the phase transition temperature. In addition to suppressing off-pathway states, β-barrel assembly machinery-catalyzed folding in vivo could accelerate any or all of these main folding steps to ensure efficient outer membrane protein biogenesis in vivo.

The Lipoprotein BamB Facilitates Folding and Insertion of Outer Membrane Protein A (OmpA) into Lipid Membranes Depending on Bilayer Thickness

Insertion and folding of β-barrel membrane proteins (β-MPs) into lipid bilayers was investigated for outer membrane protein A (OmpA). In Escherichia coli, the assembly of β-MPs is mediated by the barrel assembly machinery (BAM) complex. This complex is composed of the β-MP BamA and 4 lipoproteins, BamB, C, D, and E. We have previously examined the impact of individual BAM proteins, like BamA on insertion and folding of OmpA [1]. Here, BamB was isolated from membrane fractions of E. coli to investigate its properties. BamB folding was reversible even in the absence of lipids as examined by circular dichroism spectroscopy. BamB bound to preformed lipid bilayers of DLPC at a stoichiometry of ∼580±125. The effects of BamB alone on the kinetics of folding and insertion of OmpA were explored for lipid bilayers of different thickness. Interestingly, BamB improved the rates and yields of folding of OmpA into lipid bilayers of DLPC/DLPE/DLPG (5:3:2), but not into lipid bilayers of DOPC/DOPE/DOPG (5:3:2). This was observed for peripherally bound BamB, but not for cytochrome c, which was used for comparisons. A thorough analysis of the folding kinetics of OmpA suggests a stoichiometry of 1:1 for transient interactions of OmpA with BamB. [1] G. Patel, J.H. Kleinschmidt, Biochemistry, 52 (2013) 3974-3986.

Single-Molecule Detection of Insertion and Folding of OmpA in Droplet Interface Bilayers

The Outer Membrane Proteins of E. coli are a family of membrane-spanning beta-barrels which enable vital communication with the surrounding environment. The folding and insertion of OmpA into the membrane is the archetype for beta-barrel protein folding. Here we monitor the folding dynamics of OmpA into Droplet Interface Bilayers using single-molecule FRET. Energy transfer reports on the folded state of individual molecules imaged using TIRF microscopy. We explore the kinetics of initial binding and subsequent insertion into the bilayer.

The Lipid Bilayer-Inserted Membrane Protein BamA of Escherichia coli Facilitates Insertion and Folding of Outer Membrane Protein A from Its Complex with Skp

Folding of β-barrel membrane proteins, either from a urea-unfolded form or from chaperone-bound aqueous forms, has been characterized for pure lipid bilayers. The impact of preinserted integral proteins from biomembranes has not been examined in biophysical comparisons, but this knowledge is important for the characterization of protein assembly machinery in membranes to distinguish specific effects from unspecific effects. Here, folding was studied for a β-barrel membrane protein, outer membrane protein A (OmpA) from Escherichia coli, in the absence and presence of two other preinserted integral proteins, BamA of the β-barrel assembly machinery complex (BAM) from E. coli and FomA from Fusobacterium nucleatum. Three different preformed lipid membranes of phosphatidylcholine were prepared to compare the folding kinetics of OmpA, namely, proteoliposomes containing either BamA or FomA and pure liposomes. Urea-unfolded OmpA folded faster into phosphatidylcholine bilayers containing FomA than into pure lipid bilayers, but the kinetics of OmpA folding and insertion were fastest for bilayers containing BamA. Incorporation of BamA into lipid bilayers composed of phosphatidylcholine and phosphatidylethanolamine greatly weakened the inhibiting effect of phosphatidylethanolamine on the folding of OmpA. Folding of OmpA from its complex with the periplasmic chaperone Skp into bilayers composed of phosphatidylethanolamine and phosphatidylcholine was inhibited in the absence of BamA but facilitated when BamA was present, indicating an interaction of Skp-OmpA complexes with BamA.

Periplasmic Chaperones and their Function in the Folding of Outer Membrane Protein A into Lipid Membranes

In Gram-negative bacteria, outer membrane proteins (OMPs) are translocated across the cytoplasmic membrane in unfolded form via the Sec translocon. Prior to insertion and folding into the outer membrane, the OMPs have to traverse the periplasm. Molecular chaperones prevent OMP aggregation before the OMPs reach the outer membrane. OMPs like outer membrane protein A (OmpA) have been shown to interact with chaperones, like Skp (1-3) and SurA (4-6).OmpA folds spontaneously into lipid bilayers from a urea-unfolded form when the denaturant urea is strongly diluted. We have examined the kinetics of folding of OmpA and how it is affected by the chaperones Skp, SurA, FkpA. In all experiments, either in the absence or in the presence of any of these chaperones, the kinetics are well-described by two parallel kinetic processes of OmpA folding and insertion into lipid bilayers. Both processes were of first order. All examined chaperones increased the contribution of the faster folding process. However, only SurA caused an increase of the rate constant of the fast folding process. The temperature dependence of OmpA folding into lipid bilayers indicated that SurA lowers the activation energy of folding for the faster process.

A Kinetic Analysis of the Folding and Unfolding of OmpA in Urea and Guanidinium Chloride: Single and Parallel Pathways

The outer membrane protein OmpA from Escherichia coli can fold into lipid vesicles and surfactant micelles from the urea-denatured state. However, a complete kinetic description of the folding and unfolding of OmpA, which can provide the basis for subsequent protein engineering studies of the protein's folding pathway, is lacking. Here we use two different denaturants to probe the unfolding mechanism of OmpA in the presence of the surfactant octyl maltoside (OM). Unfolding of OmpA in the presence of micelles, achieved with the potent denaturant guanidinium chloride (GdmCl), leads to single-phase unfolding. In contrast, OmpA unfolds in urea only below OM's critical micelle concentration, and this occurs in different phases, which we attribute to the existence of states that have bound different amounts of surfactant, from completely "naked" to partly covered by surfactant. Multiple parallel refolding phases are attributed to different levels of collapse prior to folding. Kinetic results used to derive the stability of OmpA in surfactant, using either urea or GdmCl as the denaturing agent, give comparable results and indicate a minimalist three-state folding scheme involving denatured state D, folding intermediate I, and native state N. N and I are stabilized by 15.6 and 2.6 kcal/mol, respectively, relative to D. The periplasmic domain of OmpA does not contribute to stability in surfactant micelles. However, BBP, a minimalist transmembrane β-barrel version of OmpA with shortened loops, is destabilized by ~10 kcal/mol compared to OmpA, highlighting loop contributions to OmpA stability.

Effects of the Periplasmic Domain of BamA, the Essential Transmembrane Protein of the Beta-Barrel Assembly Machinery (BAM), in Folding and Insertion of the Outer Membrane Protein A

In Escherichia coli, folding and insertion of outer membrane proteins (OMPs) is facilitated by the β-barrel assembly machinery (BAM), composed of the integral protein BamA and the four lipoproteins BamB, BamC, BamA and BamE. BamA consists of a transmembrane β-barrel and a soluble periplasmic domain (PD-BamA), comprised of five polypeptide transport-associated (POTRA) domains. Although it is known that PD-BamA is necessary for the activity of the BAM-complex, its mechanism is not understood. To examine whether PD-BamA promotes folding and insertion of OMPs into lipid membranes, we expressed and isolated it from E. coli. The secondary structure of PD-BamA was determined by CD spectroscopy to 36% α-helix, 15% β-strand, 20% β-turns, and 29% random coil. For OMP folding experiments, we isolated outer membrane protein A (OmpA) from E. coli in unfolded form in urea-solution. To investigate the effect of PD-BamA on the kinetics of OmpA folding into the model membranes different sets of experiments were performed. In parallel experiments, in the absence and presence of PD-BamA, the dependence of OmpA folding was examined by gel electrophoresis as described [1]. The effects of temperature, lipid composition, and lipid concentration on PD-BamA function were investigated. PD-BamA was found to promote folding of OmpA into lipid bilayers when phosphatidylglycerol (PG) was present in the membrane. No effect on the folding of OmpA could be observed in the absence of PG. Fluorescence spectroscopy showed that PD-BamA binds to lipid bilayers, but only when these contain PG. The kinetics of OmpA folding, determined at various PD-BamA/OmpA ratios, indicated a 1:1 stoichiometry of the interaction of PD-BamA and unfolded OmpA.[1] Kleinschmidt, J.H. (2003) Cell. Mol. Life Sci. 60, 1547-1558.

Cell-free synthesis and folding of transmembrane OmpA reveals higher order structures and premature truncations

We use a cell-free transcription–translation system to monitor the effect of different lipids on the synthesis and folding of the transmembrane domain of the outer membrane protein OmpA from E. coli under physiological conditions. Folding is consistent with previous observations made in vitro at high pH. Synthesis and folding yields are optimal in phosphocholine lipids, particularly in short chain lipids and small vesicles, while lipid rafts do not promote folding compared to the folding in the absence of lipids. Truncated species are observed during translation in the presence of the periplasmic chaperone Skp, which likely binds to the newly synthesized polypeptide chain during cell-free translation and thus prematurely terminate polypeptide chain synthesis. In contrast, folded and unfolded dimers of OmpA correlate negatively with folding yields. This suggests that dimer formation competes with folding and insertion of monomeric OmpA, though folded dimers slowly appear to convert to folded monomers.

The Periplasmic Chaperone Skp Facilitates Targeting, Insertion, and Folding of OmpA into Lipid Membranes with a Negative Membrane Surface Potential

The basic biochemical and biophysical principles by which chaperone-bound membrane proteins are targeted to the outer membrane of Gram-negative bacteria for insertion and folding are unknown. Here we compare spontaneous folding of outer membrane protein A (OmpA) of Escherichia coli from its urea-unfolded form and from the complex with its periplasmic chaperone Skp into lipid bilayers. Skp facilitated folding of OmpA into negatively charged membranes containing dioleoylphosphatidylglycerol (DOPG). In contrast, Skp strongly inhibited folding of OmpA when bilayers were composed of dioleoylphosphatidylethanolamine and dioleoylphosphatidylcholine (DOPC). These results indicate that the positively charged Skp targets OmpA to a negatively charged membrane, which facilitates the release of OmpA from its complex with Skp for subsequent folding and membrane insertion. The dual functionality of Skp as a chaperone and as a targeting protein is ideal to mediate the transport of OmpA and other outer membrane proteins across the periplasm in a folding-competent form to the outer membrane, which is negatively charged on its periplasmic side. OmpA (pI 5.5) folded most efficiently above its isoelectric point. In the absence of Skp and in contrast to folding into DOPC bilayers, insertion and folding of OmpA were retarded for membranes containing DOPG at neutral or basic pH because of electrostatic repulsion. When folding of OmpA was performed near its isoelectric point, urea dilution led to a more compact aqueous form of OmpA previously characterized by fluorescence, which folded at a much slower rate. Under conditions where two different aqueous conformations of OmpA coexisted, e.g., in the titration region of OmpA, the last step of OmpA folding could be well described by two parallel pseudo-first-order kinetic phases. In this kinetic model, the contribution of the faster folding process, but not the changes in the rate constants, determined the folding yields obtained at different pH. The faster phase dominated when the experimental conditions favored the less compact form of aqueous OmpA.

Comparison of Insertion and Folding of Chaperone-bound Outer Membrane Protein A (OmpA) of E. coli into Phospholipid Bilayers of Various Composition

OmpA spontaneously inserts and folds into lipid bilayers from a urea-unfolded state upon urea-dilution. Previous work demonstrated that urea can be replaced effectively by the periplasmic chaperone Skp when lipopolysaccharide (LPS), a component of the outer membrane, is present [1]. Skp was shown to bind outer membrane proteins with nanomolar affinity and to prevent their aggregation [2].Here we investigated folding of Skp-bound OmpA into lipid bilayers of different headgroup composition and chain-length, both in absence and presence of LPS. For urea-denatured OmpA and in absence of Skp and LPS, kinetics of folding into bilayers containing the negatively charged phosphatidylglycerol showed a lag-phase of up to 30 min for dilauroylphospholipid bilayers (LUVs, 100 nm diameter) prior to folding. Skp inhibited folding and prolonged the lag-phase at basic pH when LPS was absent. In presence of LPS, no lag-phase was observed and folding rates increased dramatically.When similar experiments were performed with bilayers composed of the corresponding dioleolylphospholipids (SUVs), a lag-phase was not observed, but Skp inhibited very strongly. LPS again stimulated OmpA insertion and folding, albeit not as much as observed for dilaurylphospholipid bilayers.Skp inhibited folding also in experiments with neutral phosphatidylcholine bilayers, irrespective of lipid chain-length. Here, LPS could also facilitate folding of Skp-bound OmpA, but the effect was less pronounced for dioleoylphosphatidylcholine bilayers. The data suggest, LPS-assisted folding of Skp-bound OmpA depends on both, the surface charge of the membrane and on the lipid-chain composition.

The Trimeric Periplasmic Chaperone Skp of Escherichia coli Forms 1:1 Complexes with Outer Membrane Proteins via Hydrophobic and Electrostatic Interactions

The interactions of outer membrane proteins (OMPs) with the periplasmic chaperone Skp from Escherichia coli are not well understood. We have examined the binding of Skp to various OMPs of different origin, size, and function. These were OmpA, OmpG, and YaeT (Omp85) from Escherichia coli, the translocator domain of the autotransporter NalP from Neisseria meningitides, FomA from Fusobacterium nucleatum, and the voltage-dependent anion-selective channel, human isoform 1 (hVDAC1) from mitochondria. Binding of Skp was observed for bacterial OMPs, but neither for hVDAC1 nor for soluble bovine serum albumin. The Skp trimer formed 1:1 complexes, OMP·Skp 3, with bacterial OMPs, independent of their size or origin. The dissociation constants of these OMP·Skp 3 complexes were all in the nanomolar range, indicating that they are stable. Complexes of Skp 3 with YaeT displayed the smallest dissociation constants, complexes with NalP the largest. OMP binding to Skp 3 was pH-dependent and not observed when either Skp or OMPs were neutralized at very basic or very acidic pH. When the ionic strength was increased, the free energies of binding of Skp to OmpA or OmpG were reduced. Electrostatic interactions were therefore necessary for formation and stability of OMP·Skp 3 complexes. Light-scattering and circular dichroism experiments demonstrated that Skp 3 remained a stable trimer from pH 3 to pH 11. In the OmpA·Skp 3 complex, Skp efficiently shielded tryptophan residues of the transmembrane strands of OmpA against fluorescence quenching by aqueous acrylamide. Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, bound to OmpA·Skp 3 complexes at low stoichiometries. Acrylamide quenching of fluorescence indicated that in this ternary complex, the tryptophan residues of the transmembrane domain of OmpA were located closer to the surface than in binary OmpA·Skp 3 complexes. This may explain previous observations that folding of Skp-bound OmpA into lipid bilayers is facilitated in presence of LPS.

Folding and Insertion of the Outer Membrane Protein OmpA Is Assisted by the Chaperone Skp and by Lipopolysaccharide

We have studied the folding pathway of a beta-barrel membrane protein using outer membrane protein A (OmpA) of Escherichia coli as an example. The deletion of the gene of periplasmic Skp impairs the assembly of outer membrane proteins of bacteria. We investigated how Skp facilitates the insertion and folding of completely unfolded OmpA into phospholipid membranes and which are the biochemical and biophysical requirements of a possible Skp-assisted folding pathway. In refolding experiments, Skp alone was not sufficient to facilitate membrane insertion and folding of OmpA. In addition, lipopolysaccharide (LPS) was required. OmpA remained unfolded when bound to Skp and LPS in solution. From this complex, OmpA folded spontaneously into lipid bilayers as determined by electrophoretic mobility measurements, fluorescence spectroscopy, and circular dichroism spectroscopy. The folding of OmpA into lipid bilayers was inhibited when one of the periplasmic components, either Skp or LPS, was absent. Membrane insertion and folding of OmpA was most efficient at specific molar ratios of OmpA, Skp, and LPS. Unfolded OmpA in complex with Skp and LPS folded faster into phospholipid bilayers than urea-unfolded OmpA. Together, these results describe a first assisted folding pathway of an integral membrane protein on the example of OmpA.

Outer membrane protein A of E. coli folds into detergent micelles, but not in the presence of monomeric detergent.

Outer membrane protein A (OmpA) of Escherichia coli is a beta-barrel membrane protein that unfolds in 8 M urea to a random coil. OmpA refolds upon urea dilution in the presence of certain detergents or lipids. To examine the minimal requirements for secondary and tertiary structure formation in beta-barrel membrane proteins, folding of OmpA was studied as a function of the hydrophobic chain length, the chemical structure of the polar headgroup, and the concentration of a large array of amphiphiles. OmpA folded in the presence of detergents only above a critical minimal chain length of the apolar chain as determined by circular dichroism spectroscopy and a SDS-PAGE assay that measures tertiary structure formation. Details of the chemical structure of the polar headgroup were unimportant for folding. The minimal chain length required for folding correlated with the critical micelle concentration in each detergent series. Therefore, OmpA requires preformed detergent micelles for folding and does not adsorb monomeric detergent to its perimeter after folding. Formation of secondary and tertiary structure is thermodynamically coupled and strictly dependent on the interaction with aggregated amphiphiles.

Refolding of an integral membrane protein. OmpA of Escherichia coli.

OmpA is an integral membrane protein from the outer membrane of Escherichia coli. Purified, lipopolysaccharide-free OmpA was denatured by boiling in sodium dodecyl sulfate (SDS). Refolding was then induced by replacement of SDS with the nonionic detergent octylglucoside. The structure of both the denatured and refolded protein were investigated by SDS-gel electrophoresis, protease digestion, Raman and fluorescence spectroscopy. Refolded OmpA could be reconstituted into membranes of the synthetic lipid dimyristoylphosphatidylcholine. Thus, lipopolysaccharide is neither necessary for proper folding of OmpA nor for its insertion into lipid membranes. Based on this result, models for sorting of OmpA into the outer membrane of E. coli are discussed.