Folding Of Β-barrel Membrane Proteins Research Articles

Folding of β-Barrel Membrane Proteins into Lipid Membranes by Site-Directed Fluorescence Spectroscopy.

Protein-lipid interactions are important for folding and membrane insertion of integral membrane proteins that are composed either of α-helical or of β-barrel structure in their transmembrane domains. While α-helical transmembrane proteins fold co-translationally while they are synthesized by a ribosome, β-barrel transmembrane proteins (β-TMPs) fold and insert posttranslationally-in bacteria after translocation across the cytoplasmic membrane, in cell organelles of eukaryotes after import across the outer membrane of the organelle. β-TMPs can be unfolded in aqueous solutions of chaotropic denaturants like urea and spontaneously refold upon denaturant dilution in the presence of preformed lipid bilayers. This facilitates studies on lipid interactions during folding into lipid bilayers. For several β-TMPs, the kinetics of folding has been reported as strongly dependent on protein-lipid interactions. The kinetics of adsorption/insertion and folding of β-TMPs can be monitored by fluorescence spectroscopy. These fluorescence methods are even more powerful when combined with site-directed mutagenesis for the preparation of mutants of a β-TMP that are site-specifically labeled with a fluorophore or a fluorophore and fluorescence quencher or fluorescence resonance energy acceptor. Single tryptophan or single cysteine mutants of the β-TMP allow for the investigation of local protein-lipid interactions, at specific regions within the protein. To examine the structure formation of β-TMPs in a lipid environment, fluorescence spectroscopy has been used for double mutants of β-TMPs that contain a fluorescent tryptophan and a spin-label, covalently attached to a cysteine as a fluorescence quencher. The sites of mutation are selected so that the tryptophan is in close proximity to the quencher at the cysteine only when the β-TMP is folded. In a folding experiment, the evolution of fluorescence quenching as a function of time at specific sites within the protein can provide important information on the folding mechanism of the β-TMP. Here, we report protocols to examine membrane protein folding for two β-TMPs in a lipid environment, the outer membrane protein A from Escherichia coli, OmpA, and the voltage-dependent anion-selective channel, human isoform 1, hVDAC1, from mitochondria.

The Escherichia coli β-Barrel Assembly Machinery Is Sensitized to Perturbations under High Membrane Fluidity.

Integral β-barrel membrane proteins are folded and inserted into the Gram-negative bacterial outer membrane by the β-barrel assembly machine (BAM). This essential complex, composed of a β-barrel protein, BamA, and four lipoproteins, BamB, BamC, BamD, and BamE, resides in the outer membrane, a unique asymmetrical lipid bilayer that is difficult to recapitulate in vitro Thus, the probing of BAM function in living cells is critical to fully understand the mechanism of β-barrel folding. We recently identified an anti-BamA monoclonal antibody, MAB1, that is a specific and potent inhibitor of BamA function. Here, we show that the inhibitory effect of MAB1 is enhanced when BAM function is perturbed by either lowering the level of BamA or removing the nonessential BAM lipoproteins, BamB, BamC, or BamE. The disruption of BAM reduces BamA activity, increases outer membrane (OM) fluidity, and activates the σE stress response, suggesting the OM environment and BAM function are interconnected. Consistent with this idea, an increase in the membrane fluidity through changes in the growth environment or alterations to the lipopolysaccharide in the outer membrane is sufficient to provide resistance to MAB1 and enable the BAM to tolerate these perturbations. Amino acid substitutions in BamA at positions in the outer membrane spanning region or the periplasmic space remote from the extracellular MAB1 binding site also provide resistance to the inhibitory antibody. Our data highlight that the outer membrane environment is a critical determinant in the efficient and productive folding of β-barrel membrane proteins by BamA.IMPORTANCE BamA is an essential component of the β-barrel assembly machine (BAM) in the outer membranes of Gram-negative bacteria. We have used a recently described inhibitory anti-BamA antibody, MAB1, to identify the molecular requirements for BAM function. Resistance to this antibody can be achieved through changes to the outer membrane or by amino acid substitutions in BamA that allosterically affect the response to MAB1. Sensitivity to MAB1 is achieved by perturbing BAM function. By using MAB1 activity and functional assays as proxies for BAM function, we link outer membrane fluidity to BamA activity, demonstrating that an increase in membrane fluidity sensitizes the cells to BAM perturbations. Thus, the search for potential inhibitors of BamA function must consider the membrane environment in which β-barrel folding occurs.

Monoclonal antibody targeting the β-barrel assembly machine of Escherichia coli is bactericidal

The folding and insertion of integral β-barrel membrane proteins into the outer membrane of Gram-negative bacteria is required for viability and bacterial pathogenesis. Unfortunately, the lack of selective and potent modulators to dissect β-barrel folding in vivo has hampered our understanding of this fundamental biological process. Here, we characterize a monoclonal antibody that selectively inhibits an essential component of the Escherichia coli β-barrel assembly machine, BamA. In the absence of complement or other immune factors, the unmodified antibody MAB1 demonstrates bactericidal activity against an E. coli strain with truncated LPS. Direct binding of MAB1 to an extracellular BamA epitope inhibits its β-barrel folding activity, induces periplasmic stress, disrupts outer membrane integrity, and kills bacteria. Notably, resistance to MAB1-mediated killing reveals a link between outer membrane fluidity and protein folding by BamA in vivo, underscoring the utility of this antibody for studying β-barrel membrane protein folding within a living cell. Identification of this BamA antagonist highlights the potential for new mechanisms of antibiotics to inhibit Gram-negative bacterial growth by targeting extracellular epitopes.

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.

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.

Single-Molecule Observation of Folding and Insertion of Outer Membrane Proteins in Droplet Interface Bilayers

Outer membrane proteins (OMPs) are key players in the interface between Gram-negative bacteria and the environment. OmpA serves as an archetypical model of β-barrel membrane protein folding and insertion [1]. Here we use TIRF (Total Internal Reflection Fluorescence) and FRET (Förster Resonance Energy Transfer) microscopy in a droplet interface bilayer system [2] to investigate the folding and insertion on a single-molecule level. [1] Kleinschmidt, J.H. Cellular and Molecular Life Sciences 2003 60, 1547 [2] Thompson, J. R. et al. Nano Letters 2007 7, 3875

Physical Determinants of β-Barrel Membrane Protein Folding in Lipid Vesicles

The spontaneous folding of two Neisseria outer membrane proteins, opacity-associated (Opa) 60 and Opa 50 into lipid vesicles was investigated by systematically varying bulk and membrane properties. Centrifugal fractionation coupled with sodium dodecyl sulfate polyacrylamide gel electrophoresis mobility assays enabled the discrimination of aggregate, unfolded membrane-associated, and folded membrane-inserted protein states as well as the influence of pH, ionic strength, membrane surface potential, lipid saturation, and urea on each. Protein aggregation was reduced with increasing lipid chain length, basic pH, low salt, the incorporation of negatively charged guest lipids, or by the addition of urea to the folding reaction. Insertion from the membrane-associated form was improved in shorter chain lipids, with more basic pH and low ionic strength; it is hindered by unsaturated or ether-linked lipids. The isolation of the physical determinants of insertion suggests that the membrane surface and dipole potentials are driving forces for outer membrane protein insertion and folding into lipid bilayers.

In vitro association of fragments of a β-sheet membrane protein

Although the β-barrel membrane protein OmpA can be produced in a biologically active form in E. coli from co-expressed fragments, the fragments have not been demonstrated to associate in vitro. We have produced 3 complementary fragment pairs of OmpA which can associate to form a folded complex according to the SDS band-shift assay. We are able to convert 25–35% of the fragment populations to non-covalent but SDS-stable complexes. The periplasmic chaperone Skp effectively prevented this association. Two separately expressed and purified overlapping fragments of OmpA can form a protease-resistant complex that undergoes the characteristic band-shift upon heating. Our work demonstrates that although membrane insertion and folding of β-barrel membrane proteins may be a cooperative process, the fragments can associate in vitro without any additional components. However, the low yield and slow folding rates indicate that partially unfolded or destabilized β-sheet membrane proteins can potentially engage in many non-native interactions.

The transition state for folding of an outer membrane protein

Inspired by the seminal work of Anfinsen, investigations of the folding of small water-soluble proteins have culminated in detailed insights into how these molecules attain and stabilize their native folds. In contrast, despite their overwhelming importance in biology, progress in understanding the folding and stability of membrane proteins remains relatively limited. Here we use mutational analysis to describe the transition state involved in the reversible folding of the beta-barrel membrane protein PhoPQ-activated gene P (PagP) from a highly disordered state in 10 M urea to a native protein embedded in a lipid bilayer. Analysis of the equilibrium stability and unfolding kinetics of 19 variants that span all eight beta-strands of this 163-residue protein revealed that the transition-state structure is a highly polarized, partly formed beta-barrel. The results provide unique and detailed insights into the transition-state structure for beta-barrel membrane protein folding into a lipid bilayer and are consistent with a model for outer membrane protein folding via a tilted insertion mechanism.

Correct Folding of the β-Barrel of the Human Membrane Protein VDAC Requires a Lipid Bilayer

Spontaneous membrane insertion and folding of β-barrel membrane proteins from an unfolded state into lipid bilayers has been shown previously only for few outer membrane proteins of Gram-negative bacteria. Here we investigated membrane insertion and folding of a human membrane protein, the isoform 1 of the voltage-dependent anion-selective channel (hVDAC1) of mitochondrial outer membranes. Two classes of transmembrane proteins with either α-helical or β-barrel membrane domains are known from the solved high-resolution structures. VDAC forms a transmembrane β-barrel with an additional N-terminal α-helix. We demonstrate that similar to bacterial OmpA, urea-unfolded hVDAC1 spontaneously inserts and folds into lipid bilayers upon denaturant dilution in the absence of folding assistants or energy sources like ATP. Recordings of the voltage-dependence of the single channel conductance confirmed folding of hVDAC1 to its active form. hVDAC1 developed first β-sheet secondary structure in aqueous solution, while the α-helical structure was formed in the presence of lipid or detergent. In stark contrast to bacterial β-barrel membrane proteins, hVDAC1 formed different structures in detergent micelles and phospholipid bilayers, with higher content of β-sheet and lower content of α-helix when inserted and folded into lipid bilayers. Experiments with mixtures of lipid and detergent indicated that the content of β-sheet secondary structure in hVDAC1 decreased at increased detergent content. Unlike bacterial β-barrel membrane proteins, hVDAC1 was not stable even in mild detergents such as LDAO or dodecylmaltoside. Spontaneous folding of outer membrane proteins into lipid bilayers indicates that in cells, the main purpose of membrane-inserted or associated assembly factors may be to select and target β-barrel membrane proteins towards the outer membrane instead of actively assembling them under consumption of energy as described for the translocons of cytoplasmic membranes.

Sequence Motifs and Antimotifs in β-Barrel Membrane Proteins from a Genome-Wide Analysis: The Ala-Tyr Dichotomy and Chaperone Binding Motifs

β-barrel membrane proteins are found in the outer membrane of gram-negative bacteria, mitochondria, and chloroplasts. Although sequence motifs have been studied in α-helical membrane proteins and have been shown to play important roles in their assembly, it is not clear whether over-represented motifs and under-represented anti-motifs exist in β-barrel membrane proteins. We have developed probabilistic models to identify sequence motifs of residue pairs on the same strand separated by an arbitrary number of residues. A rigorous statistical model is essential for this study because of the difficulty associated with the short length of the strands and the small amount of structural data. By comparing to the null model of exhaustive permutation of residues within the same β-strand, propensity values of sequence patterns of two residues and p-values measuring statistical significance are calculated exactly by several analytical formulae we have developed or by enumeration. We find that there are characteristic sequence motifs and antimotifs in transmembrane (TM) β-strands. The amino acid Tyr plays an important role in several such motifs. We find a general dichotomy consisting of favorable Aliphatic-Tyr sequence motifs and unfavorable Tyr-Aliphatic antimotifs. Tyr is also part of a terminal motif, YxF, which is likely to be important for chaperone binding. Our results also suggest several experiments that can help to elucidate the mechanisms of in vitro and in vivo folding of β-barrel membrane proteins.

Folding of β-sheets in membranes: specificity and promiscuity in peptide model systems

The interactions that drive the folding of β-barrel membrane proteins have not been well studied because there have been few available model systems for membrane β-sheets. In this work, we expand on a recently described model system to explore the contributions of interstrand hydrogen bonds, side-chain/side-chain interactions and side-chain/membrane interactions to β-sheet formation in membranes. These experiments are based on the observation that the hydrophobic hexapeptide acetyl-Trp-Leu-Leu-Leu-Leu-Leu-OH (AcWLLLLL) folds, cooperatively and reversibly, into oligomeric, antiparallel β-sheets in phosphatidylcholine membranes. To systematically characterize the important interactions that drive β-sheet formation in membranes, we have used circular dichroism spectroscopy to determine the membrane secondary structure of each member of a complete host-guest family of related peptides of the form AcWLL-X-LL, where X is one of the natural amino acids. Peptides with hydrophobic X-residues of any size or character (X = Ala, Val, Ile, Leu, Cys, Met, Phe and Trp) form similar β-sheets in membranes, while peptides with any polar X-residue or Gly or Pro at the X-position are random-coils, even when bound to membranes at high concentrations. The observed membrane sheet preferences correlate poorly with intrinsic sheet propensity scales measured in soluble proteins, but they correlate well with several membrane hydrophobicity scales. These results support the idea that the predominant interactions of the side-chains in membrane-bound β-sheets are with the membrane lipids, and that backbone hydrogen bonding is the major driving force for the stabilization of β-sheets in membranes.