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The insertion of membrane proteins requires proteinaceous complexes in the cytoplasm, the membrane, and the lumen of organelles. Most of the required complexes have been described, while the components for insertion of β‐barrel‐type proteins into the outer membrane of chloroplasts remain unknown. The same holds true for the signals required for the insertion of β‐barrel‐type proteins. At present, only the processing of Toc75‐III, the β‐barrel‐type protein of the central chloroplast translocon with an atypical signal, has been explored in detail. However, it has been debated whether Toc75‐V/ outer envelope protein 80 (OEP80), a second protein of the same family, contains a signal and undergoes processing. To substantiate the hypothesis that Toc75‐V/OEP80 is processed as well, we reinvestigated the processing in a protoplast‐based assay as well as in native membranes. Our results confirm the existence of a cleavable segment. By protease protection and pegylation, we observed intermembrane space localization of the soluble N‐terminal domain. Thus, Toc75‐V contains a cleavable N‐terminal signal and exposes its polypeptide transport‐associated domains to the intermembrane space of plastids, where it likely interacts with its substrates.

Escherichia coli periplasmic zinc-metallopeptidase BepA normally functions by promoting maturation of LptD, a β-barrel outermembrane protein involved in biogenesis of lipopolysaccharides, but degrades it when its membrane assembly is hampered. These processes should be properly regulated to ensure normal biogenesis of LptD. The underlying mechanism of regulation, however, remains to be elucidated. A recently solved BepA structure has revealed unique features: In particular, the active site is buried in the protease domain and conceivably inaccessible for substrate degradation. Additionally, the His-246 residue in the loop region containing helix α9 (α9/H246 loop), which has potential flexibility and covers the active site, coordinates the zinc ion as the fourth ligand to exclude a catalytic water molecule, thereby suggesting that the crystal structure of BepA represents a latent form. To examine the roles of the α9/H246 loop in the regulation of BepA activity, we constructed BepA mutants with a His-246 mutation or a deletion of the α9/H246 loop and analyzed their activities in vivo and in vitro. These mutants exhibited an elevated protease activity and, unlike the wild-type BepA, degraded LptD that is in the normal assembly pathway. In contrast, tethering of the α9/ H246 loop repressed the LptD degradation, which suggests that the flexibility of this loop is important to the exhibition of protease activity. Based on these results, we propose that the α9/ H246 loop undergoes a reversible structural change that enables His-246-mediated switching (histidine switch) of its protease activity, which is important for regulated degradation of stalled/misassembled LptD. [ABSTRACT FROM AUTHOR]

The insertion of membrane proteins requires proteinaceous complexes in the cytoplasm, the membrane, and the lumen of organelles. Most of the required complexes have been described, while the components for insertion of β‐barrel‐type proteins into the outer membrane of chloroplasts remain unknown. The same holds true for the signals required for the insertion of β‐barrel‐type proteins. At present, only the processing of Toc75‐III, the β‐barrel‐type protein of the central chloroplast translocon with an atypical signal, has been explored in detail. However, it has been debated whether Toc75‐V/ outer envelope protein 80 (OEP80), a second protein of the same family, contains a signal and undergoes processing. To substantiate the hypothesis that Toc75‐V/OEP80 is processed as well, we reinvestigated the processing in a protoplast‐based assay as well as in native membranes. Our results confirm the existence of a cleavable segment. By protease protection and pegylation, we observed intermembrane space localization of the soluble N‐terminal domain. Thus, Toc75‐V contains a cleavable N‐terminal signal and exposes its polypeptide transport‐associated domains to the intermembrane space of plastids, where it likely interacts with its substrates. [ABSTRACT FROM AUTHOR]

β-barrel membrane proteins populate the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts, playing significant roles in multiple key cellular pathways. Characterizing the functions of these membrane proteins in vivo is often challenging due to the complex protein network in the periplasm of Gram-negative bacteria (or intermembrane space in mitochondria and chloroplasts) and the presence of other outer membrane proteins. In vitro reconstitution into lipid-bilayer-like environments such as nanodiscs or proteoliposomes provides an excellent method for examining the specific function and mechanism of these membrane proteins in an isolated system. Here, we describe the methodologies employed to investigate Slam, a 14-stranded β-barrel membrane protein also known as the type XI secretion system that is responsible for translocating proteins across the outer membrane of many bacterial species., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Total interference reflection fluorescence (TIRF) microscopy of lipid bilayers is an effective technique for studying the lateral movement and ion channel activity of single integral membrane proteins. Here we describe how to integrate the mitochondrial outer membrane preprotein translocase TOM-CC and its β-barrel protein-conducting channel Tom40 into supported lipid bilayers to identify possible relationships between movement and channel activity. We propose that our approach can be readily applied to membrane protein channels where transient tethering to either membrane-proximal or intramembrane structures is accompanied by a change in channel permeation., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

ABSTRACT Proteins of the Omp85 family chaperone the membrane insertion of β-barrel-shaped outer membrane proteins in bacteria, mitochondria, and probably chloroplasts and facilitate the transfer of nuclear-encoded cytosolically synthesized preproteins across the outer envelope of chloroplasts. This protein family is characterized by N-terminal polypeptide transport-associated (POTRA) domains and a C-terminal membrane-embedded β-barrel. We have investigated a recently identified Omp85 family member of Arabidopsis thaliana annotated as P39. We show by in vitro and in vivo experiments that P39 is localized in chloroplasts. The electrophysiological properties of P39 are consistent with those of other Omp85 family members confirming the sequence based assignment of P39 to this family. Bioinformatic analysis showed that P39 lacks any POTRA domain, while a complete 16 stranded β-barrel including the highly conserved L6 loop is proposed. The electrophysiological properties are most comparable to Toc75-V, which is consistent with the phylogenetic clustering of P39 in the Toc75-V rather than the Toc75-III branch of the Omp85 family tree. Taken together P39 forms a pore with Omp85 family protein characteristics. The bioinformatic comparison of the pore region of Toc75-III, Toc75-V, and P39 shows distinctions of the barrel region most likely related to function. Proteins 2017; 85:1391-1401. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The voltage-dependent anion-selective channel (VDAC) is an intrinsic β-barrel membrane protein located within the mitochondrial outer membrane where it serves as a pore, connecting the mitochondria to the cytosol. The high-resolution structures of both the human and murine VDACs have been resolved by X-ray diffraction and nuclear magnetic resonance spectroscopy (NMR) in 2008. However, the structural data are not completely in line with the findings that were obtained after decades of research on biochemical and functional analysis of VDAC. This discrepancy may be related to the fact that structural biology studies of membrane proteins reveal specific static conformations that may not necessarily represent the physiological state. For example, overexpression of membrane proteins in bacterial inclusion bodies or simply the extraction from the native lipid environment using harsh purification methods ( i.e . chaotropic agents) can disturb the physiological conformations and the supramolecular assemblies. To address these potential issues, we have developed a method, allowing rapid one step purification of endogenous VDAC expressed in the native mitochondrial membrane without overexpression of recombinant protein or usage of harsh chaotropic extraction procedures. Using the Saccharomyces cerevisiae isoform 1 of VDAC as a model, this method yields efficient purification, preserving VDAC in a more physiological, native state following extraction from mitochondria. Single particle analysis using transmission electron microscopy (TEM) demonstrated conservation of oligomeric assembly after purification. Maintenance of the native state was evaluated using functional assessment that involves an ATP-binding assay by micro-scale thermophoresis (MST). Using this approach, we were able to determine for the first time the apparent K D for ATP of 1.2 mM. [ABSTRACT FROM AUTHOR]

Gram-negative bacteria contain a double membrane which serves for both protection and for providing nutrients for viability. The outermost of these membranes is called the outer membrane (OM), and it contains a host of fully integrated membrane proteins which serve essential functions for the cell, including nutrient uptake, cell adhesion, cell signalling and waste export. For pathogenic strains, many of these outer membrane proteins (OMPs) also serve as virulence factors for nutrient scavenging and evasion of host defence mechanisms. OMPs are unique membrane proteins in that they have a β-barrel fold and can range in size from 8 to 26 strands, yet can still serve many different functions for the cell. Despite their essential roles in cell survival and virulence, the exact mechanism for the biogenesis of these OMPs into the OM has remained largely unknown. However, the past decade has witnessed significant progress towards unravelling the pathways and mechanisms necessary for moulding a nascent polypeptide into a functional OMP within the OM. Here, we will review some of these recent discoveries that have advanced our understanding of the biogenesis of OMPs in Gram-negative bacteria, starting with synthesis in the cytoplasm to folding and insertion into the OM. [ABSTRACT FROM AUTHOR]

Proteins of the Omp85 family are involved in the insertion of β-barrel shaped outer membrane proteins in bacteria and mitochondria, and-at least-in the transfer of preproteins across the chloroplast outer envelope. In general these proteins consist of up to five N-terminal 'polypeptide transport associated' (POTRA) domains and a C-terminal, membrane embedded β-barrel domain. In Arabidopsis thaliana two plastidic gene families coding for Omp85-like proteins exist, namely the Toc75-III and the Toc75-V/Oep80 sub-family. The latter is composed of three genes, of which two do not contain POTRA domains. These are annotated as P39 and P36. However, P36 resulted from a very recent gene duplication of P39 and appears to be specific to Arabidopsis thaliana. Furthermore, we show that P39 is specifically expressed in vein tissues, while P36 is expressed at early and late developmental stages. T-DNA insertion in P36 causes a mild phenotype with reduced starch accumulation in chloroplasts of sepals pointing towards a yet to be described plastid function. [ABSTRACT FROM AUTHOR]

Abstract: Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes in the RND (resistance–nodulation–cell division) family to expel diverse toxic compounds from the cell. These complexes span both the inner and outer membranes of the bacterium via an α-helical, inner membrane transporter; a periplasmic membrane fusion protein; and a β-barrel, outer membrane channel. One such efflux system, CusCBA, is responsible for extruding biocidal Cu(I) and Ag(I) ions. To remove these toxic ions, the CusC outer membrane channel must form a β-barrel structural domain, which creates a pore and spans the entire outer membrane. We here report the crystal structures of wild-type CusC, as well as two CusC mutants, suggesting that the first N-terminal cysteine residue plays an important role in protein–membrane interactions and is critical for the insertion of this channel protein into the outer membrane. These structures provide insight into the mechanisms on CusC folding and transmembrane channel formation. It is found that the interactions between CusC and membrane may be crucial for controlling the opening and closing of this β-barrel, outer membrane channel. [Copyright &y& Elsevier]

Abstract: We report structural studies in aqueous solution on backbone cyclic peptides that possess potent antimicrobial activity specifically against Pseudomonas sp. The peptides target the β-barrel outer membrane protein LptD, which plays an essential role in lipopolysaccharide transport to the outer membrane. The peptide L27-11 contains a 12-residue loop (T1W2L3K4K5R6R7W8K9K10A11K12) linked to a DPro–LPro template. Two related peptides were also studied, one with various Lys to ornithine or diaminobutyric acid substitutions as well as a DLys6 (called LB-01), and another containing the same loop sequence, but linked to an LPro–DPro template (called LB-02). NMR studies and MD simulations show that L27-11 and LB-01 adopt β-hairpin structures in solution. In contrast, LB-02 is more flexible and importantly, adopts a wide variety of different backbone conformations, but not β-hairpin conformations. L27-11 and LB-01 show antimicrobial activity in the nanomolar range against Pseudomonas aeruginosa , whereas LB-02 is essentially inactive. Thus the β-hairpin structure of the peptide is important for antimicrobial activity. An alanine scan of L27-11 revealed that tryptophan side chains (W2/W8) displayed on opposite faces of the β-hairpin represent key groups contributing to antimicrobial activity. [Copyright &y& Elsevier]

Membrane proteins in detergent micelles are large and dynamic complexes that present challenges for solution NMR investigations such as spectral overlap and line broadening. In this study, multiple methods are introduced to facilitate resonance assignment of β-barrel membrane proteins using Opa60 from Neisseria gonorrhoeae as a model system. Opa60 is an eight-stranded β-barrel with long extracellular loops (∼63% of the protein) that engage host receptors and induce engulfment of the bacterium. The NMR spectra of Opa60 in detergent micelles exhibits significant spectral overlap and resonances corresponding to the loop regions had variable line widths, which interfered with a complete assignment of the protein. To assign the β-barrel residues, trypsin cleavage was used to remove much of the extracellular loops while preserving the detergent solubilized β-barrel. The removal of the loop resonances significantly improved the assignment of the Opa60 β-barrel region (97% of the resonances corresponding to the β-barrel and periplasmic turns were assigned). For the loop resonance assignments, two strategies were implemented; modulating temperature and synthetic peptides. Lowering the temperature broadened many peaks beyond detection and simplified the spectra to only the most dynamic regions of the loops facilitating 27 loop resonances to be assigned. To further assign functionally important and unstructured regions of the extracellular loops, a synthetic 20 amino acid peptide was synthesized and had nearly complete spectral overlap with the full-length protein allowing 17 loop resonances to be assigned. Collectively, these strategies are effective tools that may accelerate solution NMR structure determination of β-barrel membrane proteins. [ABSTRACT FROM AUTHOR]

Abstract: The pH dependent opening and closure of Escherichia coli OmpG is driven by the formation and breaking of hydrogen bridges in β-strands S11–S13. We have investigated the in situ secondary structural changes of OmpG with ATR–FTIR difference spectroscopy in order to detect the signals associated with the newly established interactions. Curve-fitting of OmpG in two pH conditions revealed the splitting and shifting of β-sheet signals upon opening of the channel. Besides secondary structure changes, there are also amino acid side chain signals that play active role in opening/closing of the channel. An interaction among positively charged arginines and negatively charged aspartic and glutamic acid residues is suggested upon closure of the channel while this interaction is abolished when the channel opens at higher pH. [Copyright &y& Elsevier]

Abstract: Escherichia coli BamB is the largest of four lipoproteins in the β-barrel assembly machinery (BAM) complex. It interacts with the periplasmic domain of BamA, an integral outer membrane protein (OMP) essential for OMP biogenesis. Although BamB is not essential, it serves an important function in the BAM complex, significantly increasing the folding efficiency of some OMPs in vivo and in vitro. To learn more about the BAM complex, we solved structures of BamB in three different crystal forms. BamB crystallized in space groups P213, I222, and P212121, with one molecule per asymmetric unit in each case. Crystals from the space group I222 diffracted to 1. 65-Å resolution. BamB forms an eight-bladed β-propeller with a central pore and is shaped like a doughnut. A DALI search revealed that BamB shares structural homology to several eukaryotic proteins containing WD40 repeat domains, which commonly have β-propeller folds and often serve as scaffolding proteins within larger multi-protein complexes that carry out signal transduction, cell division, and chemotaxis. Using mutagenesis data from previous studies, we docked BamB onto a BamA structural model and assessed known and possible interactions between these two proteins. Our data suggest that BamB serves as a scaffolding protein within the BAM complex by optimally orienting the flexible periplasmic domain of BamA for interaction with other BAM components and chaperones. This may facilitate integration of newly synthesized OMPs into the outer membrane. [Copyright &y& Elsevier]

Abstract: Background: Hidden Markov models (HMMs) have been extensively used in computational molecular biology, for modelling protein and nucleic acid sequences. The design of the model architecture and the algorithms for parameter estimation and decoding are extremely important for improve the performance of HMM. In topology prediction of transmembrane β-barrels proteins (TMBs), the Baum–Welch algorithm is widely adapted for HMM training but usually leads to a sub-optimal model in practice. In addition, all the existing HMM-based predictors are only designed to model the transmembrane segment without a submodel to model the signal peptide (SP) for full-length sequences. It is not convenient for users to investigate the structures of full-length TMB sequences. Results: We present here, an HMM that combine a transmembrane barrel submodel and an SP submodel for both topology and SP predictions. A new genetic algorithm (GA) is presented here to training the model, at the same time the Posterior–Viterbi algorithm is adopted for decoding. A dataset including 33 TMBs that is the most so far in literature are collected for model training and testing. Results of self-consistency and jackknife tests shows the GA has better global performance than the Baum–Welch algorithm. Results of jackknife tests show that this method performs better than all well known existing methods for topology predictions. Furthermore, it provides a function to predict SP in full-length TMBs sequences with fairish accuracy. Conclusion: We show that our combined HMM-based method is a better choice for TMB topology prediction, which implements topology predictions with higher accuracy and additional SP predictions for full-length TMB sequences. [Copyright &y& Elsevier]

Abstract: The physical interactions that switch the functional state of membrane proteins are poorly understood. Previously, the pH-gating conformations of the β-barrel forming outer membrane protein G (OmpG) from Escherichia coli have been solved. When the pH changes from neutral to acidic the flexible extracellular loop L6 folds into and closes the OmpG pore. Here, we used single-molecule force spectroscopy to structurally localize and quantify the interactions that are associated with the pH-dependent closure. At acidic pH, we detected a pH-dependent interaction at loop L6. This interaction changed the (un)folding of loop L6 and of β-strands 11 and 12, which connect loop L6. All other interactions detected within OmpG were unaffected by changes in pH. These results provide a quantitative and mechanistic explanation of how pH-dependent interactions change the folding of a peptide loop to gate the transmembrane pore. They further demonstrate how the stability of OmpG is optimized so that pH changes modify only those interactions necessary to gate the transmembrane pore. [Copyright &y& Elsevier]

Abstract: A gating mechanism of the β-barrel-forming outer membrane protein G (OmpG) from Escherichia coli was recently presented. The mechanism was based on X-ray structures revealed from crystals grown from solubilized OmpG at both neutral pH and acidic pH. To investigate whether these conformations represent the naturally occurring gating mechanism, we reconstituted OmpG in native E. coli lipids and applied high-resolution atomic force microscopy. The reconstituted OmpG molecules assembled into both monomers and dimers. Single monomeric and dimeric OmpG molecules showed open channel entrances at pH 7.5 and at room temperature. The extracellular loops connecting the β-strands that form the transmembrane β-barrel pore exhibited elevated structural flexibility. Upon lowering the pH to 5.0, the conformation of OmpG molecules changed to close the extracellular entrance of their channel. It appears that one or more of the extracellular loops collapsed onto the channel entrance. This conformational change was fully reversible. Our data confirm that the previously reported gating mechanism of OmpG occurs at physiological conditions in E. coli lipid membranes. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

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 β-barrel that is predicted to consist of 14 β-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 1.1nS at 1M KCl, in agreement with the conductance of FomA isolated from membranes in native form. In contrast to OmpA, which forms a smaller eight-stranded β-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 β-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 19kJ/mol (dicapryl phosphatidylcholine) and 70kJ/mol (dioleoyl phosphatidylcholine) for the faster pathways. [Copyright &y& Elsevier]

β-Barrel membrane proteins are found in the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts. Little is known about how residues in membrane β-barrels interact preferentially with other residues on adjacent strands. We have developed probabilistic models to quantify propensities of residues for different spatial locations and for interstrand pairwise contact interactions involving strong H-bonds, side-chain interactions, and weak H-bonds. Using the reference state of exhaustive permutation of residues within the same β-strand, the propensity values and p-values measuring statistical significance are calculated exactly by analytical formulae we have developed. Our findings show that there are characteristic preferences of residues for different membrane locations. Contrary to the “positive-inside” rule for helical membrane proteins, β-barrel membrane proteins follow a significant albeit weaker “positive-outside” rule, in that the basic residues Arg and Lys are disproportionately favored in the extracellular cap region and disfavored in the periplasmic cap region. We find that different residue pairs prefer strong backbone H-bonded interstrand pairings (e.g. Gly–aromatic) or non-H-bonded pairings (e.g. aromatic–aromatic). In addition, we find that Tyr and Phe participate in aromatic rescue by shielding Gly from polar environments. We also show that these propensities can be used to predict the registration of strand pairs, an important task for the structure prediction of β-barrel membrane proteins. Our accuracy of 44% is considerably better than random (7%). It also significantly outperforms a comparable registration prediction for soluble β-sheets under similar conditions. Our results imply several experiments that can help to elucidate the mechanisms of in vitro and in vivo folding of β-barrel membrane proteins. The propensity scales developed in this study will also be useful for computational structure prediction and for folding simulations. [Copyright &y& Elsevier]

Escherichia coli periplasmic zinc-metallopeptidase BepA normally functions by promoting maturation of LptD, a β-barrel outer-membrane protein involved in biogenesis of lipopolysaccharides, but degrades it when its membrane assembly is hampered. These processes should be properly regulated to ensure normal biogenesis of LptD. The underlying mechanism of regulation, however, remains to be elucidated. A recently solved BepA structure has revealed unique features: In particular, the active site is buried in the protease domain and conceivably inaccessible for substrate degradation. Additionally, the His-246 residue in the loop region containing helix α9 (α9/H246 loop), which has potential flexibility and covers the active site, coordinates the zinc ion as the fourth ligand to exclude a catalytic water molecule, thereby suggesting that the crystal structure of BepA represents a latent form. To examine the roles of the α9/H246 loop in the regulation of BepA activity, we constructed BepA mutants with a His-246 mutation or a deletion of the α9/H246 loop and analyzed their activities in vivo and in vitro. These mutants exhibited an elevated protease activity and, unlike the wild-type BepA, degraded LptD that is in the normal assembly pathway. In contrast, tethering of the α9/H246 loop repressed the LptD degradation, which suggests that the flexibility of this loop is important to the exhibition of protease activity. Based on these results, we propose that the α9/H246 loop undergoes a reversible structural change that enables His-246-mediated switching (histidine switch) of its protease activity, which is important for regulated degradation of stalled/misassembled LptD., Competing Interests: The authors declare no competing interest.

Proteins of the Omp85 family chaperone the membrane insertion of β-barrel-shaped outer membrane proteins in bacteria, mitochondria, and probably chloroplasts and facilitate the transfer of nuclear-encoded cytosolically synthesized preproteins across the outer envelope of chloroplasts. This protein family is characterized by N-terminal polypeptide transport-associated (POTRA) domains and a C-terminal membrane-embedded β-barrel. We have investigated a recently identified Omp85 family member of Arabidopsis thaliana annotated as P39. We show by in vitro and in vivo experiments that P39 is localized in chloroplasts. The electrophysiological properties of P39 are consistent with those of other Omp85 family members confirming the sequence based assignment of P39 to this family. Bioinformatic analysis showed that P39 lacks any POTRA domain, while a complete 16 stranded β-barrel including the highly conserved L6 loop is proposed. The electrophysiological properties are most comparable to Toc75-V, which is consistent with the phylogenetic clustering of P39 in the Toc75-V rather than the Toc75-III branch of the Omp85 family tree. Taken together P39 forms a pore with Omp85 family protein characteristics. The bioinformatic comparison of the pore region of Toc75-III, Toc75-V, and P39 shows distinctions of the barrel region most likely related to function. Proteins 2017; 85:1391-1401. © 2014 Wiley Periodicals, Inc., (© 2014 Wiley Periodicals, Inc.)

In cells, β-barrel membrane proteins are transported in unfolded form to an outer membrane into which they fold and insert. Model systems have been established to investigate the mechanisms of insertion and folding of these versatile proteins into detergent micelles, lipid bilayers and even synthetic amphipathic polymers. In these experiments, insertion into lipid membranes is initiated from unfolded forms that do not display residual β-sheet secondary structure. These studies therefore have allowed the investigation of membrane protein folding and insertion in great detail. Folding of β-barrel membrane proteins into lipid bilayers has been monitored from unfolded forms by dilution of chaotropic denaturants that keep the protein unfolded as well as from unfolded forms present in complexes with molecular chaperones from cells. This review is aimed to provide an overview of the principles and mechanisms observed for the folding of β-barrel transmembrane proteins into lipid bilayers, the importance of lipid-protein interactions and the function of molecular chaperones and folding assistants. This article is part of a Special Issue entitled: Lipid-protein interactions., (Copyright © 2015 Elsevier B.V. All rights reserved.)

β-barrel outer membrane proteins (OMPs) are found in the outer membranes (OMs) of all gram-negative bacteria, yet exactly how they are folded and inserted remains unknown. The last decade has provided a wealth of discovery including the identification of the BAM complex, a multicomponent complex responsible for the biogenesis of all OMPs into the OM. It is anticipated that the next decade will further advance our knowledge of how the BAM complex is able to perform its unique and interesting function.

The utility of protein engineering, both the mutation and deletion of specific amino acids, to investigate protein structure and function has been demonstrated time and time again, and intermolecular and intramolecular interactions within the BAM complex and its individual components are no exception. Extensive efforts have probed conserved and unique amino acid sequences of the Bam proteins to define their functional roles. This chapter summarizes efforts as applied to the disulfide cross-link mutants of BamA and describes experimental methods used in our studies to determine that lateral opening of the barrel domain is required for function.

β-barrel membrane proteins are somewhat unique in that their folding states can be monitored using semi-native SDS-PAGE methods to determine if they are folded properly or not. This property, which is commonly referred to as heat modifiability, has been used for many years on both purified protein and on whole cells to monitor folded states of proteins of interest. Additionally, heat modifiability assays have proven indispensable in studying the BAM complex and its role in folding and inserting β-barrel membrane proteins into the outer membrane. Here, we describe the protocol our lab uses for performing the heat modifiability assay in our studies on outer membrane proteins.

Membrane proteins in detergent micelles are large and dynamic complexes that present challenges for solution NMR investigations such as spectral overlap and line broadening. In this study, multiple methods are introduced to facilitate resonance assignment of β-barrel membrane proteins using Opa60 from Neisseria gonorrhoeae as a model system. Opa60 is an eight-stranded β-barrel with long extracellular loops (∼63% of the protein) that engage host receptors and induce engulfment of the bacterium. The NMR spectra of Opa60 in detergent micelles exhibits significant spectral overlap and resonances corresponding to the loop regions had variable line widths, which interfered with a complete assignment of the protein. To assign the β-barrel residues, trypsin cleavage was used to remove much of the extracellular loops while preserving the detergent solubilized β-barrel. The removal of the loop resonances significantly improved the assignment of the Opa60 β-barrel region (97% of the resonances corresponding to the β-barrel and periplasmic turns were assigned). For the loop resonance assignments, two strategies were implemented; modulating temperature and synthetic peptides. Lowering the temperature broadened many peaks beyond detection and simplified the spectra to only the most dynamic regions of the loops facilitating 27 loop resonances to be assigned. To further assign functionally important and unstructured regions of the extracellular loops, a synthetic 20 amino acid peptide was synthesized and had nearly complete spectral overlap with the full-length protein allowing 17 loop resonances to be assigned. Collectively, these strategies are effective tools that may accelerate solution NMR structure determination of β-barrel membrane proteins., (© 2013 The Protein Society.)