Transmembrane Β-barrel Proteins Research Articles

Recombinant Expression and Overproduction of Transmembrane β-Barrel Proteins.

Transmembrane β-barrel proteins reside in the outer membrane of Gram-negative bacteria and are thus in direct contact with the environment. Because of that, they are involved in many key processes stretching from cellular survival to virulence. Hence, they are an attractive target for the development of novel antimicrobials, in addition to being of fundamental biological interest. To study this class of proteins, they are often required to be expressed in Escherichia coli. Recombinant expression of β-barrel proteins can be achieved using two fundamentally different strategies. The first alternative uses a complete coding sequence that includes a signal peptide for targeting the protein to its native cellular location, the bacterial outer membrane. The second alternative omits the signal peptide in the gene, leading to mislocalization and aggregation of the protein in the bacterial cytoplasm. These aggregates, called inclusion bodies, can be solubilized and the protein can be folded into its native form in vitro. In this chapter, we present example protocols for both strategies and discuss their advantages and disadvantages.

Analysis of Transmembrane β-Barrel Proteins by Native and Semi-native Polyacrylamide Gel Electrophoresis.

Membrane-embedded β-barrel proteins are important regulators of the outer membrane permeability barrier of Gram-negative bacteria. β-barrels are highly structured domains formed by a series of antiparallel β-strands. Each β-strand is locked in position by hydrogen bonds between its polypeptide backbone andthose of thetwo neighbouring strands in the barrel structure. Some transmembraneβ-barrel proteins form larger homo- or hetero-multimeric complexes that accomplish specific functions. In this chapter, we describe native and semi-native polyacrylamide gel electrophoresis (PAGE) methods to characterize the organization of transmembraneβ-barrel proteins. We illustrate blue native (BN)-PAGE as an analytical method to assess the formation of protein complexes. Furthermore, we describe a heat-modifiability assay via semi-native PAGE as arapid method to investigate the folding of transmembrane β-barrels.

Inhibitors targeting BamA in gram-negative bacteria

Antibiotic resistance has led to an increase in the number of patient hospitalizations and deaths. The situation for gram-negative bacteria is especially dire as the last new class of antibiotics active against these bacteria was introduced to the clinic over 60 years ago, thus there is an immediate unmet need for new antibiotic classes able to overcome resistance. The outer membrane, a unique and essential structure in gram-negative bacteria, contains multiple potential antibacterial targets including BamA, an outer membrane protein that folds and inserts transmembrane β-barrel proteins. BamA is essential and conserved, and its outer membrane location eliminates a barrier that molecules must overcome to access this target. Recently, antibacterial small molecules, natural products, peptides, and antibodies that inhibit BamA activity have been reported, validating the druggability of this target and generating potential leads for antibiotic development. This review will describe these BamA inhibitors, highlight their key attributes, and identify challenges with this potential target.

Comparing the dynamics of the BAM complex between two species

Outer membrane proteins (OMPs) in Gram-negative bacteria are transmembrane β-barrel proteins that are involved in important biological activities such as nutrient transport, signal transduction and the export of virulence factors. While it’s known that the protein complex responsible for assembly and insertion of OMPs into the outer membrane is the β-barrel assembly machinery (BAM), the detailed mechanisms for OMP insertion by BAM are only recently coming into focus. Nonetheless, to date, the only complete high-resolution structures of BAM are from Escherichia coli, leaving open the question of the degree of conservation of mechanisms across species.

OMPdb: A Global Hub of Beta-Barrel Outer Membrane Proteins.

OMPdb (www.ompdb.org) was introduced as a database for β-barrel outer membrane proteins from Gram-negative bacteria in 2011 and then included 69,354 entries classified into 85 families. The database has been updated continuously using a collection of characteristic profile Hidden Markov Models able to discriminate between the different families of prokaryotic transmembrane β-barrels. The number of families has increased ultimately to a total of 129 families in the current, second major version of OMPdb. New additions have been made in parallel with efforts to update existing families and add novel families. Here, we present the upgrade of OMPdb, which from now on aims to become a global repository for all transmembrane β-barrel proteins, both eukaryotic and bacterial.

De novo design of transmembrane β barrels.

Transmembrane β-barrel proteins (TMBs) are of great interest for single-molecule analytical technologies because they can spontaneously fold and insert into membranes and form stable pores, but the range of pore properties that can be achieved by repurposing natural TMBs is limited. We leverage the power of de novo computational design coupled with a "hypothesis, design, and test" approach to determine TMB design principles, notably, the importance of negative design to slow β-sheet assembly. We design new eight-stranded TMBs, with no homology to known TMBs, that insert and fold reversibly into synthetic lipid membranes and have nuclear magnetic resonance and x-ray crystal structures very similar to the computational models. These advances should enable the custom design of pores for a wide range of applications.

Building a barrel

Protein Design Computational design offers the possibility of making proteins with customized structures and functions. The range of accessible protein scaffolds has expanded with the design of increasingly complex cytoplasmic proteins and, recently, helical membrane proteins. Vorobieva et al. describe the successful computational design of eight-stranded transmembrane β-barrel proteins (TMBs). Using an iterative approach, they show the importance of negative design to prevent off-target structures and gain insight into the sequence determinants of TMB folding. Twenty-three designs satisfied biochemical screens for a TMB structure, and two structures were experimentally validated by nuclear magnetic resonance spectroscopy or x-ray crystallography. This is a step toward the custom design of pores for applications such as single-molecule sequencing. Science , this issue p. [eabc8182][1] [1]: /lookup/doi/10.1126/science.abc8182

Antigenic outer membrane proteins prediction of Pasteurella multocida serotype B:2

Outer membrane proteins (OMPs) are one of the prominent virulence factor or immunogenic element of Pasteurella multocida which are responsible for eliciting immune responses in multiple infected hosts. Identification of these proteins allows researchers to target OMPs to be manipulated as a vaccine against bacterial infection. Precise and rapid bioinformatics tools allow researchers to perform in silico analysis to extract putative OMPs from the genome information. In this study, we have successfully identified 105 putative OMPs of P. multocida subsp. multocida strain PMTB2.1 through computational prediction tools including a subcellular localisation predictor, PSORTb v3.0 followed by a lipoprotein predictor, LipoP 1.0 and a β-barrel transmembrane protein predictor, BOMP for sub-classification of the OMPs into 53 integral and 52 peripheral OMPs of this strain. The manipulation of antigenic epitope predictors and the antigenicity score filtering identified nine putative antigenic OMPs. These putative predicted antigenic OMPs of this pathogen will provide crucial initial guidance for the experimental identification and selection of antigenic protein(s) for the development of future haemorrhagic septicaemia (HS) vaccine.

Chloroplast Outer Membrane β-Barrel Proteins Use Components of the General Import Apparatus.

Chloroplasts evolved from a cyanobacterial endosymbiont that resided within a eukaryotic cell. Due to their prokaryotic heritage, chloroplast outer membranes contain transmembrane β-barrel proteins. While most chloroplast proteins use N-terminal transit peptides to enter the chloroplasts through the translocons at the outer and inner chloroplast envelope membranes (TOC/TIC), only one β-barrel protein, Toc75, has been shown to use this pathway. The route other β-barrel proteins use has remained unresolved. Here we use in vitro pea (Pisum sativum) chloroplast import assays and transient expression in Nicotiana benthamiana to address this. We show that a paralog of Toc75, outer envelope protein 80 kD (OEP80), also uses a transit peptide but has a distinct envelope sorting signal. Our results additionally indicate that β-barrels that do not use transit peptides also enter the chloroplast using components of the general import pathway.

Kinetics of Insertion and Folding of Outer Membrane Proteins by Gel Electrophoresis.

To examine the mechanisms of folding and insertion of TMPs into membranes, kinetic studies are instrumental, for example, for the analysis of folding steps and involved intermediates or for the determination of activation energies. For many β-barrel transmembrane proteins (β-TMPs) it has been shown that the folded, functional form can be separated from the unfolded form by a simple electrophoretic mobility assay. The only requirements for a separation by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) are that the folded form is sufficiently stable and that the samples are not heat-denatured before the electrophoresis is performed. Many folded β-TMPs resist the treatment with SDS at room temperature and are stable against forces during electrophoresis. On the other side, SDS also binds to unfolded forms of β-TMPs and prevents their folding into β-barrel structure. These observations have been used to develop a simple assay to monitor the kinetics of β-barrel tertiary structure formation in a membrane environment by electrophoresis. A folding reaction of a β-TMP is initiated by dilution of the denaturant in the presence of preformed lipid bilayers, proteoliposomes or membrane vesicles. At selected times, samples are taken from the reaction. In these samples, folding is stopped by addition of SDS. At the end of the entire folding reaction, all samples are analyzed by SDS-PAGE and the fractions of folded β-TMP that they contain are determined by densitometry.An advantage of this kinetic assay is that it not only allows a direct determination of fractions of folded and unfolded forms at a selected time during folding of the β-TMP into a membrane, but also facilitates the determination of the impact of folding factors (e.g., molecular chaperones) or folding machinery that most often have a different molecular mass and electrophoretic mobility. The assay has been very useful to examine how folding and insertion is affected by the structure of the phospholipids in the lipid bilayer and how folding machinery compensates for the presence of membrane lipids that retard folding and insertion of β-TMPs.

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.

β-Strand twisting/bending in soluble and transmembrane β-barrel structures.

The majority of β-strands in globular proteins have a right-handed twist and bend. The dominant driving force for β-strand twisting is thought to be inter-strand hydrogen bonds. We previously demonstrated that for water-soluble proteins, both the twisting and bending of β-strand are suppressed by the polar side chains of serine, threonine, and asparagine residues. To determine whether this also holds for transmembrane β-strands, we calculated and statistically analyzed the twist and bend angles of four-residue frames of β-strands in both transmembrane and water-soluble β-barrel proteins with known three-dimensional structures. In the case of transmembrane β-strands, we found that twisting was suppressed even for frames not containing serine, threonine, or asparagine residues. The suppression of twisting in transmembrane β-strands could be attributed to the propagation of the suppressive effect of serine, threonine, and asparagine residues within a frame to the neighboring, hydrogen-bonded strands under the restriction that all strands in the closed barrel structure must have similar twist angles. A similar tendency was also observed for water-soluble β-barrel proteins. We previously showed that the dominant driving force for β-strand bending is hydrophobic interactions involving aromatic residues within and outside the strand. Transmembrane β-barrels have no hydrophobic core; however, rather hydrophilic residues predominate inside the barrel or the β-strands of transmembrane β-barrels have larger bend angles than those of water-soluble β-barrels. Our results reveal that, in transmembrane β-barrel proteins, the glycine-aromatic ring motif is important for generating the β-strand bending necessary for barrel formation.

C-terminal kink formation is required for lateral gating in BamA

In Gram-negative bacteria, the outer membrane contains primarily β-barrel transmembrane proteins and lipoproteins. The insertion and assembly of β-barrel outer-membrane proteins (OMPs) is mediated by the β-barrel assembly machinery (BAM) complex, the core component of which is the 16-stranded transmembrane β-barrel BamA. Recent studies have indicated a possible role played by the seam between the first and last β-barrel strands of BamA in the OMP insertion process through lateral gating and a destabilized membrane region. In this study, we have determined the stability and dynamics of the lateral gate through over 12.5 μs of equilibrium simulations and 4 μs of free-energy calculations. From the equilibrium simulations, we have identified a persistent kink in the C-terminal strand and observed spontaneous lateral-gate separation in a mimic of the native bacterial outer membrane. Free-energy calculations of lateral gate opening revealed a significantly lower barrier to opening in the C-terminal kinked conformation; mutagenesis experiments confirm the relevance of C-terminal kinking to BamA structure and function.

Single-Molecule Force Spectroscopy of Transmembrane β-Barrel Proteins.

Single-molecule force spectroscopy (SMFS) has been widely applied to study the mechanical unfolding and folding of transmembrane proteins. Here, we review the recent progress in characterizing bacterial and human transmembrane β-barrel proteins by SMFS. First, we describe the mechanical unfolding of transmembrane β-barrels, which follows a general mechanism dictated by the sequential unfolding and extraction of individual β-strands and β-hairpins from membranes. Upon force relaxation, the unfolded polypeptide can insert stepwise into the membrane as single β-strands or β-hairpins to fold as the native β-barrel. The refolding can be followed at a high spatial and temporal resolution, showing that small β-barrels are able to fold without assistance, whereas large and complex β-barrels require chaperone cofactors. Applied in the dynamic mode, SMFS can quantify the kinetic and mechanical properties of single β-hairpins and reveal complementary insight into the membrane protein structure and function relationship. We further outline the challenges that SMFS experiments must overcome for a comprehensive understanding of the folding and function of transmembrane β-barrel proteins.

Folding Determinants of Transmembrane β-Barrels Using Engineered OMP Chimeras.

Transmembrane β-barrel proteins (OMPs) are highly robust structures for engineering and development of nanopore channels, surface biosensors, and display libraries. Expanding the applications of designed OMPs requires the identification of elements essential for β-barrel scaffold formation and stability. Here, we have designed chimeric 8-stranded OMPs composed of strand hybrids of Escherichia coli OmpX and Yersinia pestis Ail, and identified molecular motifs essential for β-barrel scaffold formation. For the OmpX/Ail chimeras, we find that the central hairpin strands β4-β5 in tandem are vital for β-barrel folding. We also show that the central hairpin can facilitate OMP assembly even when present as the N- or C-terminal strands. Further, the C-terminal β-signal and strand length are important but neither sufficient nor mutually exclusive for β-barrel assembly. Our results point to a nonstochastic model for assembly of chimeric β-barrels in lipidic micelles. The assembly likely follows a predefined nucleation at the central hairpin only when presented in tandem, with some influence from its absolute position in the barrel. Our findings can lead to the design of engineered barrels that retain the OMP assembly elements necessary to attain well-folded, stable, yet malleable scaffolds, for bionanotechnology applications.

Energetics of side-chain partitioning of β-signal residues in unassisted folding of a transmembrane β-barrel protein

The free energy of water-to-interface amino acid partitioning is a major contributing factor in membrane protein folding and stability. The interface residues at the C terminus of transmembrane β-barrels form the β-signal motif required for assisted β-barrel assembly in vivo but are believed to be less important for β-barrel assembly in vitro. Here, we experimentally measured the thermodynamic contribution of all 20 amino acids at the β-signal motif to the unassisted folding of the model β-barrel protein PagP. We obtained the partitioning free energy for all 20 amino acids at the lipid-facing interface (ΔΔG0w,i(φ)) and the protein-facing interface (ΔΔG0w,i(π)) residues and found that hydrophobic amino acids are most favorably transferred to the lipid-facing interface, whereas charged and polar groups display the highest partitioning energy. Furthermore, the change in non-polar surface area correlated directly with the partitioning free energy for the lipid-facing residue and inversely with the protein-facing residue. We also demonstrate that the interface residues of the β-signal motif are vital for in vitro barrel assembly, because they exhibit a side chain–specific energetic contribution determined by the change in nonpolar accessible surface. We further establish that folding cooperativity and hydrophobic collapse are balanced at the membrane interface for optimal stability of the PagP β-barrel scaffold. We conclude that the PagP C-terminal β-signal motif influences the folding cooperativity and stability of the folded β-barrel and that the thermodynamic contributions of the lipid- and protein-facing residues in the transmembrane protein β-signal motif depend on the nature of the amino acid side chain.

Discovery of Novel Leptospirosis Vaccine Candidates Using Reverse and Structural Vaccinology.

Leptospira spp. are diderm (two membranes) bacteria that infect mammals causing leptospirosis, a public health problem with global implications. Thousands of people die every year due to leptospirosis, especially in developing countries with tropical climates. Prophylaxis is difficult due to multiple factors, including the large number of asymptomatic hosts that transmit the bacteria, poor sanitation, increasing numbers of slum dwellers, and the lack of an effective vaccine. Several leptospiral recombinant antigens were evaluated as a replacement for the inactivated (bacterin) vaccine; however, success has been limited. A prospective vaccine candidate is likely to be a surface-related protein that can stimulate the host immune response to clear leptospires from blood and organs. In this study, a comprehensive bioinformatics approach based on reverse and structural vaccinology was applied toward the discovery of novel leptospiral vaccine candidates. The Leptospira interrogans serovar Copenhageni strain L1-130 genome was mined in silico for the enhanced identification of conserved β-barrel (βb) transmembrane proteins and outer membrane (OM) lipoproteins. Orthologs of the prospective vaccine candidates were screened in the genomes of 20 additional Leptospira spp. Three-dimensional structural models, with a high degree of confidence, were created for each of the surface-exposed proteins. Major histocompatibility complex II (MHC-II) epitopes were identified, and their locations were mapped on the structural models. A total of 18 βb transmembrane proteins and 8 OM lipoproteins were identified. These proteins were conserved among the pathogenic Leptospira spp. and were predicted to have epitopes for several variants of MHC-II receptors. A structural and functional analysis of the sequence of these surface proteins demonstrated that most βb transmembrane proteins seem to be TonB-dependent receptors associated with transportation. Other proteins identified included, e.g., TolC efflux pump proteins, a BamA-like OM component of the βb transmembrane protein assembly machinery, and the LptD-like LPS assembly protein. The structural mapping of the immunodominant epitopes identified the location of conserved, surface-exposed, immunogenic regions for each vaccine candidate. The proteins identified in this study are currently being evaluated for experimental evidence for their involvement in virulence, disease pathogenesis, and physiology, in addition to vaccine development.

Simulations of Bama in a Native Outer-Membrane Model and Energetics of Lateral Opening

Gram-negative bacteria possess two cell membranes. The outer membrane is host to almost exclusively β-barrel transmembrane proteins. The β-barrel assembly machinery (BAM) is a five-protein complex responsible for the insertion and assembly of outer membrane β-barrel proteins. The first crystal structures of the transmembrane β-barrel domain of BamA were released in 2013. In these initial crystal structures, the BamA β-barrel possessed a weak interface between its first (1) and last (16) β strands, which led to strand separation within 1 μs in our equilibrium simulations. This strand separation has been proposed to act as a lateral gate for substrate passage into the membrane. In addition, a hydrophobic mismatch near the barrel seam was shown to destabilize the membrane, potentially acting to aid integration of the substrate. Furthermore, full BAM complex crystal structures were recently released, showing accessory proteins rallying around this putative insertion region using the periplasmic domains of BamA as a scaffold. In order to address remaining questions surrounding the role that BamA plays in the insertion and assembly process, we have carried out additional equilibrium simulations of BamA in several membrane bilayers. We also performed a calculation of energetic landscapes associated with lateral gate formation under various conditions. These calculations reveal a lower energetic barrier to strand separation for BamA as compared to FhaC, a member of the same (Omp85) family. They also reveal a significant role played by the C-terminal kink feature in modulating the barrier to strand separation. Finally, equilibrium simulations of NgBamA demonstrate lateral gate opening at 340K in a native lipopolysaccharide bilayer for the first time.

Polyglycine Acts as a Rejection Signal for Protein Transport at the Chloroplast Envelope.

PolyGly is present in many proteins in various organisms. One example is found in a transmembrane β-barrel protein, translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75). Toc75 requires its N-terminal extension (t75) for proper localization. t75 comprises signals for chloroplast import (n75) and envelope sorting (c75) in tandem. n75 and c75 are removed by stromal processing peptidase and plastidic type I signal peptidase 1, respectively. PolyGly is present within c75 and its deletion or substitution causes mistargeting of Toc75 to the stroma. Here we have examined the properties of polyGly-dependent protein targeting using two soluble passenger proteins, the mature portion of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (mSS) and enhanced green fluorescent protein (EGFP). Both t75-mSS and t75-EGFP were imported into isolated chloroplasts and their n75 removed. Resultant c75-mSS was associated with the envelope at the intermembrane space, whereas c75-EGFP was partially exposed outside the envelope. Deletion of polyGly or substitution of tri-Ala for the critical tri-Gly segment within polyGly caused each passenger to be targeted to the stroma. Transient expression of t75-EGFP in Nicotiana benthamiana resulted in accumulation of c75-EGFP exposed at the surface of the chloroplast, but the majority of the EGFP passenger was found free in the cytosol with most of its c75 attachment removed. Results of circular dichroism analyses suggest that polyGly within c75 may form an extended conformation, which is disrupted by tri-Ala substitution. These data suggest that polyGly is distinct from a canonical stop-transfer sequence and acts as a rejection signal at the chloroplast inner envelope.

Faltung und Einbau von Außenmembran proteinen in Lipidmembranen

β-barrel transmembrane proteins are present in outer membranes of bacteria and organelles of eukaryotic cells like mitochondria and chloroplasts. After their synthesis in the cytosol, bacterial outer membrane proteins are translocated in unfolded form across the cytoplasmic membrane into the periplasm. Chaperones assist their transport to the β-barrel assembly machinery BAM of the outer membrane. Biophysical studies on outer membrane protein A indicated a concerted folding and insertion mechanism into lipid bilayers for which the activation energy is lowered by the BAM-complex.