Engineering the protein folding landscape in gram-negative bacteria

Gram-negative bacteria, especially Escherichia coli, are often the preferred hosts for recombinant protein production because of their fast doubling times, ability to grow to high cell density, propensity for high recombinant protein titers and straightforward protein purification techniques. The utility of simple bacteria in such studies continues to improve as a result of an ever-increasing body of knowledge regarding their native protein biogenesis machinery. From translation on the ribosome to interaction with cytosolic accessory factors to transport across the inner membrane into the periplasmic space, cellular proteins interact with many different types of cellular machinery and each interaction can have a profound effect on the protein folding process. This review addresses key aspects of cellular protein folding, solubility and expression in E. coli with particular focus on the elegant biological machinery that orchestrates the transition from nascent polypeptide to folded, functional protein. Specifically highlighted are a variety of different techniques to intentionally alter the folding environment of the cell as a means to understand and engineer intracellular protein folding and stability.     

Distance Measurement on an Endogenous Membrane Transporter in E. coli Cells and Native Membranes Using EPR Spectroscopy

Membrane proteins may be influenced by the environment, and they may be unstable in detergents or fail to crystallize. As a result, approaches to characterize structures in a native environment are highly desirable. Here, we report a novel general strategy for precise distance measurements on outer membrane proteins in whole Escherichia coli cells and isolated outer membranes. The cobalamin transporter BtuB was overexpressed and spin‐labeled in whole cells and outer membranes and interspin distances were measured to a spin‐labeled cobalamin using pulse EPR spectroscopy. A comparative analysis of the data reveals a similar interspin distance between whole cells, outer membranes, and synthetic vesicles. This approach provides an elegant way to study conformational changes or protein–protein/ligand interactions at surface‐exposed sites of membrane protein complexes in whole cells and native membranes, and provides a method to validate outer membrane protein structures in their native environment.     

Solid-state NMR on a large multidomain integral membrane protein: the outer membrane protein assembly factor BamA

Multidomain proteins constitute a large part of prokaryotic and eukaryotic proteomes and play fundamental roles in various physiological processes. However, their structural characterization is challenging because of their large size and intrinsic flexibility. We show here that motional-filtered high-resolution solid-state NMR (ssNMR) experiments allow for the observation and structural analysis of very large multidomain membrane proteins that are characterized by different motional time scales. This approach was used to probe the folding of the 790-residue membrane protein BamA, which is the core component of the Escherichia coli outer membrane protein assembly machinery. A combination of dipolar- and scalar-based two-dimensional ssNMR experiments applied to two uniformly (13)C,(15)N-labeled BamA variants revealed characteristic secondary structure elements and distinct dynamics within the BamA transmembrane protein segment and the periplasmic POTRA domains. This approach hence provides a general strategy for collecting atomic-scale structural information on multidomain (membrane) proteins in a native-like environment.     

Some aspects of the active transport of amino acids

Active transport is the transport of substances against their activity gradients. In the cell, metabolic processes provide the energy for active transport through the membrane, during which amino acids are coupled with a carrier system located in the membrane. Some of the proteins involved in the translocation of amino acids have now been isolated from E. coli.     

Asymmetric droplet interface bilayers

In cell membranes, the lipid compositions of the inner and outer leaflets differ. Therefore, a robust model system that enables single-channel electrical recording with asymmetric bilayers would be very useful. We and others recently developed the droplet interface bilayer (DIB), which is formed by connecting lipid monolayer-encased aqueous droplets submerged in an oil-lipid mixture. Here, we incorporate lipid vesicles of different compositions into aqueous droplets and immerse them in an oil bath to form asymmetric DIBs (a-DIBs). Both alpha-helical and beta-barrel membrane proteins insert readily into a-DIBs, and their activity can be measured by single-channel electrical recording. We show that the gating behavior of outer membrane protein G (OmpG) from Escherichia coli differs depending on the side of insertion in an asymmetric DIB with a positively charged leaflet opposing a negatively charged leaflet. The a-DIB system provides a general platform for studying the effects of bilayer leaflet composition on the behavior of ion channels and pores.     

Membrane proteome in Escherichia coli probed by MS3 mass spectrometry: a preliminary report

Characterization of the membrane proteome is particularly intriguing since a better knowledge in this field might lead to new insights into the function of different membrane systems. Despite the biological relevance of surface proteins however, their characterization still remains a challenging task. Outer membrane proteins (OMPs) of Gram-negative bacteria are key molecules that interface the cell with the environment. Hence, surface proteins of Gram-negative bacteria contain proteins that might be good targets for drugs, antimicrobials or detection systems and they may become components of effective vaccines. In this respect, Escherichia coli has been chosen as a model organism for several structural and functional studies aimed at understanding the biophysical and biochemical organization of proteins in Gram-negative cell walls. Here we present first results for the identification of bacterial surface exposed proteins in E. coli K12 based on the use of dansyl chloride labelling coupled with bidimensional tandem mass spectrometry exploiting the advantage of precursor ion/MS3 scan modes. This procedure resulted in a promising, simple, and rapid strategy for the identification of membrane proteins in E. coli as model organism, thus avoiding time-consuming procedures based on two-dimensional liquid chromatography and electrophoresis. The proteins identified could be grouped into five major families: outer membrane (29 proteins), lipoproteins (6 proteins), transmembrane (43 proteins) families.     

L-Arabinose transport and the L-arabinose binding protein of Escherichia coli.

The active accumulation of L-arabinose by arabinose induced cultures of Escherichia coli is mediated by 2 independent transport mechanisms. One, specified by the gene locus araE, is membrane bound and possesses a relatively "low affinity". The other, specified in part by the genetic locus araF, contains as a functional component the L-arabinose binding protein and functions with a "high affinity" for the substrate. The L-arabinose binding protein has been purified, partially characterized, crystallized, and sequenced.     

EFFECTS OF ACRIDINE PLUS NEAR-ULTRAVIOLET LIGHT ON THE OUTER MEMBRANE OF ESCHERICHIA COLI

Abstract The hydrophobic photosensitizer acridine plus near-ultraviolet light damages both plasma membranes and outer membranes in Escherichia coli. Two lines of evidence are presented that outer membrane proteins are affected by acridine plus near-ultraviolet light treatment and that the effect is selective for certain proteins. First, analysis of outer membrane proteins on sodium dodecylsulfate polyacrylamide gels revealed that some protein bands are diminished upon treatment while others remain unaltered. New bands also appear, suggesting degradation or crosslinking reactions. Second, bacteriophage adsorption studies showed that treatment of E. coli F cells with acridine plus near-ultraviolet light causes a loss in functionality of the receptor for phage T5. Treatment of E. coli ABU57 cells under comparable conditions has no discernable effect on the functionality of the receptor for phage BF23.     

Low 13C-background for NMR-based studies of ligand binding using 13C-depleted glucose as carbon source for microbial growth: 13C-labeled glucose and 13C-forskolin binding to the galactose-H+ symport protein GalP in Escherichia coli

Obtrusive 13C-backgrounds can be a problem in 13C NMR-based studies of ligand binding to bacterial membrane transport proteins in their natural state in inner membranes. This is largely solved for the bacterial galactose-H+ symport protein GalP by growing the producing organism Escherichia coli on 13C-depleted glucose (13C 相似文献   

Inherent antibacterial activity of a peptide-based beta-hairpin hydrogel

Among several important considerations for implantation of a biomaterial, a main concern is the introduction of infection. We have designed a hydrogel scaffold from the self-assembling peptide, MAX1, for tissue regeneration applications whose surface exhibits inherent antibacterial activity. In experiments where MAX1 gels are challenged with bacterial solutions ranging in concentrations from 2 x 10(3) colony forming units (CFUs)/dm2 to 2 x 10(9) CFUs/dm2, gel surfaces exhibit broad-spectrum antibacterial activity. Results show that the hydrogel surface is active against Gram-positive (Staphylococcus epidermidis, Staphylococcus aureus, and Streptococcus pyogenes) and Gram-negative (Klebsiella pneumoniae and Escherichia coli) bacteria, all prevalent in hospital settings. Live-dead assays employing laser scanning confocal microscopy show that bacteria are killed when they engage the surface. In addition, the surface of MAX1 hydrogels was shown to cause inner and outer membrane disruption in experiments that monitor the release of beta-galactosidase from the cytoplasm of lactose permease-deficient E. coli ML-35. These data suggest a mechanism of antibacterial action that involves membrane disruption that leads to cell death upon cellular contact with the gel surface. Although the hydrogel surface exhibits bactericidal activity, co-culture experiments indicate hydrogel surfaces show selective toxicity to bacterial versus mammalian cells. Additionally, gel surfaces are nonhemolytic toward human erythrocytes, which maintain healthy morphologies when in contact with the surface. These material attributes make MAX1 gels attractive candidates for use in tissue regeneration, even in nonsterile environments.     

Two-dimensional electrophoresis and peptide mass fingerprinting of bacterial outer membrane proteins.

Many bacterial outer membrane proteins (OMPs) are missing from two-dimensional (2-D) gel proteome maps. Recently, we developed a technique for 2-D electrophoresis (2-DE) of Escherichia coli OMPs using alkaline pH incubation for isolation of OMPs, followed by improved solubilization conditions for array by 2-DE using immobilized pH gradients. In this report, we expanded our study, examining protein components from the outer membranes of two enteric bacteria, Salmonella typhimurium and Klebsiella pneumoniae (also known as Klebsiella aerogenes), as well as the unrelated, free-living alpha-proteobacteria Caulobacter crescentus. Patterns of OMPs expression appeared remarkably conserved between members of the Enterobacteriaceae, while C. crescentus was unique, displaying a greater number of clusters of higher-molecular-weight proteins (>80 kDa). Peptide mass fingerprinting (PMF) was used for protein identification, and despite matching across-species boundaries, proved useful for first-pass protein assignment of enteric OMPs. In contrast, identification of C. crescentus OMPs was successful only when searching against its recently completed genome. For all three microorganisms examined, the majority of proteins identified on the 2-D gel appear localized to the outer membrane, a result consistent with our previous finding in Escherichia coli. In addition, we discuss some of the benefits and limitations of PMF in cross-species searching.     

Construction of Copper Removing Bacteria Through the Integration of Two-Component System and Cell Surface Display

Synthetic biological systems are becoming more and more feasible for commercial and medical purposes through the genetic engineering of several components. The simple assembly of a genetic circuit was shown to stimulate the removal of copper by bacteria through the engineering of a two-component system. The CusSR two-component systems is a regulator of Escherichia coli copper homeostatic system. In this system, genetic circuits of CusSR were fused to a cell surface display system for metal adsorption; this system is suitable for the display of a copper binding peptide through outer membrane protein C (OmpC). E. coli ompC codes for an outer membrane pore protein (porin) are induced at high osmolarity and temperature, which can also be used as an anchoring motif to accept the passenger proteins. The bacteria that produce the chimeric OmpC containing the copper binding peptide adsorbed maximum concentrations of 92.2 μmol of Cu(2+)/gram dry weight of bacterial cells. This synthetic bacterial system senses the specific heavy metal and activates a cell surface display system that acts to remove the metal.     

Isolation of Escherichia coli inner membranes by metal affinity two-phase partitioning

As reduction of sample complexity is a central issue in membrane proteomic research, the need for new pre-fractionation methods is significant. Here we present a method for fast and efficient enrichment of Escherichia coli inner membranes expressing a His-tagged integral membrane L-fucose-proton symporter (FucP). An enriched inner membrane fraction was obtained from a crude membrane mixture using affinity two-phase partitioning in combination with nickel-nitrilotriacetic acid (Ni-NTA) immobilized on agarose beads. Due to interaction between the beads and FucP, inner membranes were selectively partitioned to the bottom phase of a polymer/polymer aqueous two-phase system consisting of poly(ethylene glycol) (PEG) and dextran. The partitioning of membranes was monitored by assaying the activity of an inner membrane marker protein and measuring the total protein content in both phases. The enrichment of inner membrane proteins in the dextran phase was also investigated by proteomic methodology, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), trypsin digestion and liquid chromatography in combination with tandem mass spectrometry (LC-MS/MS). Using a high level of significance (99.95%) in the subsequent database search, 36 proteins assigned to the inner membrane were identified in the bottom phase, compared to 29 when using the standard sucrose gradient centrifugation method for inner membrane isolation. Furthermore, metal affinity two-phase partitioning was up to 10 times faster than sucrose gradient centrifugation. The separation conditions in these model experiments provide a basis for the selective isolation of E. coli membranes expressing His-tagged proteins and can therefore facilitate research on such membrane proteomes.     

Effects of copper on dielectric properties of E. coli cells

Dielectric properties of E. coli cells before and after Cu²⁺ incubation were investigated by using the dielectric spectroscopic technique. The dielectric spectra are analyzed theoretically by means of the extended three-shell ellipsoidal model, which can reflect the complicated morphological structure of E. coli cell including the outer membrane, the periplasmic space, the inner membrane and the cytoplasm. The results showed that dielectric properties of these cellular components were changed with Cu²⁺ treatment in a time- and concentration-dependent way. The permttivity of the outer membrane increased with the incubation time and concentration of Cu²⁺, possibly because polarizability of the outer leaflet of lipopolysaccharides was affected by Cu²⁺. The conductivity of the periplasmic space decreased with the incubation time and concentration of Cu²⁺, possibly due to the damage of peptidoglycan. The decreased permittivity of the inner membrane may be caused by disturbance of the lipid bilayer structure produced by Cu²⁺ incubation. The decreased cytoplasmic conductivity may be the consequence of the leakage of K⁺ from it. The cytoplasmic permittivity decreased with Cu²⁺ treatment probably because of the leakage of its some components.     

Crystallographic and spectroscopic evidence for high affinity binding of FeEDTA(H2O)- to the periplasmic nickel transporter NikA

Because nickel is both essential and toxic to a great variety of organisms, its detection and transport is highly regulated. In Escherichia coli and other related Gram-negative bacteria, high affinity nickel transport depends on proteins expressed by the nik operon. A central actor of this process is the periplasmic NikA transport protein. A previous structural report has proposed that nickel binds to NikA as a pentahydrate species. However, both stereochemical considerations and X-ray absorption spectroscopic results are incompatible with that interpretation. Here, we report the 1.8 A resolution structure of NikA and show that it binds FeEDTA(H2O)- with very high affinity. In addition, we provide crystallographic evidence that a metal-EDTA complex was also bound to the previously reported NikA structure. Our observations strongly suggest that nickel transport in E. coli requires the binding of this metal ion to a metallophore that bears significant resemblance to EDTA. They also provide a basis for the potential use of NikA in the bioremediation of toxic transition metals and the design of artificial metalloenzymes.     

Cluster phase chemistry: collisions of vibrationally excited cationic dicarboxylic acid clusters with water molecules initiate dissociation of cluster components

A homologous series of cationic gas-phase clusters of dicarboxylic acids (oxalic acid, malonic acid, succinic acid, glutaric acid, and adipic acid) generated via electrospray ionization (ESI) are investigated using collision-induced dissociation (CID). Singly charged cationic clusters with the composition (Na(+))(2n+1)(dicarboxylate(2-))(n), where n = 1-5, are observed as major gas-phase species. Significant abundances of singly charged sodiated hydrogen dicarboxylate clusters with the composition (Na(+))(2n)(dicarboxylate(2-))(n)(H+), where n = 1-6, are observed with oxalic acid, malonic acid, and succinic acid. Isolation of the clusters followed by CID results mainly in sequential loss of disodium dicarboxylate moieties for the clusters of succinic acid, glutaric acid, and adipic acid. However, the dimer of sodiated hydrogen succinate, all malonate clusters, and all oxalate clusters, with the exception of the dimer, exhibit complex chemical reactions initiated by the collision of vibrationally excited clusters with water molecules. Generally, water molecules serve as proton donors for reacting dicarboxylate anions in the cluster, initiating dissociation pathways such as the decomposition of the malonate ion to yield an acetate ion and CO(2). The reactivity of several mixed dicarboxylate clusters is also reported. For example, malonate anion is shown to be more reactive than oxalate anion for decarboxylation when both are present in a cluster. The energetics of several representative cluster phase reactions are evaluated using computational modeling. The present results for cationic clusters are compared and contrasted to earlier studies of anionic sodiated dicarboxylic acid clusters.     

Cluster phase chemistry: gas-phase reactions of anionic sodium salts of dicarboxylic acid clusters with water molecules

A homologous series of anionic gas-phase clusters of dicarboxylic acids (oxalic acid, malonic acid, succinic acid, glutaric acid, and adipic acid) generated via electrospray ionization (ESI) are investigated using collision-induced dissociation (CID). Sodiated clusters with the composition (Na(+))(2)(n+1)(dicarboxylate(2-)(n+1) for singly charged anionic clusters, where n = 1-4, are observed as major gas-phase species. Isolation of the clusters followed by CID results mainly in sequential loss of disodium dicarboxylate moieties for the clusters of succinic acid, glutaric acid, and adipic acid (C4-C6). However, all oxalate (C2) and malonate (C3) clusters and dimers (n = 1) of succinate (C4) and glutarate (C5) exhibit more complex chemistry initiated by collision of the activated cluster with water molecules. For example, with water addition, malonate clusters dissociate to yield sodium acetate, carbon dioxide, and sodium hydroxide. More generally, water molecules serve as proton donors for reacting dicarboxylate anions in the cluster and introduce energetically favorable dissociation pathways not otherwise available. Density functional theory (DFT) calculations of the binding energy of the cluster correlate well with the cluster phase reactions of oxalate and malonate clusters. Clusters of larger dicarboxylate ions (C4-C6) are more weakly bound, facilitating the sequential loss of disodium dicarboxylate moieties. The more strongly bound small dicarboxylate anions (oxalate and malonate) preferentially react with water molecules rather than dissociate to lose disodium dicarboxylate monomers when collisionally activated. Implications of these results for the atmospheric aerosol chemistry of dicarboxylic acids are discussed.     

Understanding the lid movements of LolA in Escherichia coli using molecular dynamics simulation and in silico point mutation

The Lol system in Escherichia coli is involved in localization of lipoproteins and hence is essential for growth of the organism. LolA is a periplasmic chaperone that binds to outer-membrane specific lipoproteins and transports them from inner membrane to outer membrane through LolB. The hydrophobic lipid-binding cavity of LolA consists of α-helices which act as a lid in regulating the transfer of lipoproteins from LolA to LolB. The current study aims to investigate the structural changes observed in LolA during the transition from open to closed conformation in the absence of lipoprotein. Molecular dynamics (MD) simulations were carried out for two LolA crystal structures; LolA(R43L), and in silico mutated MsL43R for a simulation time of 50 ns in water environment. We have performed an in silico point mutation of leucine to arginine in MsL43R to evaluate the importance of arginine to induce structural changes and impact the stability of protein structure. A complete dynamic analysis of open to closed conformation reveals the existence of two distinct levels; closing of lid and closing of entrance of hydrophobic cavity. Our analysis reveals that the structural flexibility of LolA is an important factor for its role as a periplasmic chaperone.     

Sequential mechanism of assembly of multidrug efflux pump AcrAB-TolC

Multidrug efflux pumps adversely affect both the clinical effectiveness of existing antibiotics and the discovery process to find new ones. In this study, we reconstituted and characterized by surface plasmon resonance the assembly of AcrAB-TolC, the archetypal multidrug efflux pump from Escherichia coli. We report that the periplasmic AcrA and the outer membrane channel TolC assemble high-affinity complexes with AcrB transporter independently from each other. Antibiotic novobiocin and MC-207,110 inhibitor bind to the immobilized AcrB but do not affect interactions between components of the complex. In contrast, DARPin inhibits interactions between AcrA and AcrB. Mutational opening of TolC channel decreases stability of interactions and promotes disassembly of the complex. The conformation of the membrane proximal domain of AcrA is critical for the formation of AcrAB-TolC and could be targeted for the development of new inhibitors.     

Functional organization of the outer membrane of escherichia coli: phage and colicin receptors as components of iron uptake systems.

The functional interaction of outer membrane proteins of E. coli can be studied using phage and colicin receptors which are essential components of penetration systems. The uptake of ferric iron in the form of the ferrichrome complex requires the ton A and ton B functions in the outer membrane of E. coli. The ton A gene product is the receptor protein for phage T5 and is required together with the ton B function by the phages T1 and ?80 to infect cells and by colicin M and the antibiotic albomycin, a structural analogue of ferrichrome, to kill cells. The ton B function is necessary for the uptake of ferric iron complexed by citrate. Iron complexed by enterochelin is only transported in the presence of the ton B and feu functions. Cells which have lost the feu function are resistant to the colicins B, I or V while ton B mutants are resistant to all 3 colicins. The interaction of the ton A, ton B, and feu functions apparently permits quite different "substrates" to overcome the permeability barrier of the outer membrane. It was shown for ferrichrome dependent iron uptake that the complexing agent was not altered and could be used repeatedly. Only very low amounts of 3H-labeled ferrichrome were found in the cell. It is possible that the iron is mobilized in the membrane and that desferri-ferrichrome is released into the medium without having entered the cytoplasm. Growth on ferrichrome as the sole iron source was used to select revertants of T5 resistant ton A mutants. All revertants exhibited wild-type properties with the exception of partial revertants. In these 4 strains, as in the ton A mutants, the ton A protein was not detectable by SDS polyacrylamide gel electrophoreses of outer membranes. Albomycin resistant mutants were selected and shown to fall into 5 categories: 1) ton A; 2) ton B mutants; 3) mutants with no iron transport defects and normal ton A/ton B functions, which might be target site mutants; 4) mutants which were deficient in ferrichrome-mediated iron uptake but had normal ton A/ton B functions. We tentatively consider that the defect might be located in the active transport system of the cytoplasmic membrane; 5) a variety of mutants with the following general properties: most of them were resistant to colicin M, transported iron poorly, and, like ton B mutants, contained additional proteins in the outer membrane. The outer membrane protein patterns of wild-type and ton B mutant strains were compared by slab gel electrophoresis in an attempt to identify a ton B protein. It was observed that under most growth conditions, ton B mutants overproduced 3 proteins of molecular weights 74,000-83,000. In extracted, iron-deficient medium, both the wild-type and ton B mutant strains had similar large amounts of these proteins in their outer membranes. The appearance of these proteins was suppressed by excess iron in both wild-type and mutant. From this evidence it is apparent that the proteins appear as a response to low intracellular iron rather than being controlled by the ton B gene...