Single channel analysis of recombinant major outer membrane protein porins from Chlamydia psittaci and Chlamydia pneumoniae

We recently demonstrated that the major outer membrane protein of Chlamydia psittaci, the primary vaccine candidate for combating chlamydial infections, functions as a porin-like ion channel. In this study, we have cloned, expressed and functionally reconstituted recombinant major outer membrane proteins from C. psittaci and Chlamydia pneumoniae and analysed them at the single channel level. Both form porin-like ion channels that are functionally similar to those formed by native C. psittaci major outer membrane protein. Also, like the native channels, recombinant C. psittaci channels are modified by a native major outer membrane protein-specific monoclonal antibody. This is the first time that native function has been demonstrated for recombinant chlamydial major outer membrane proteins. Future bilayer reconstitution will provide a strategy for detailed structure/function studies of this new subclass of bacterial porins and the work also has important implications for successful protein refolding and the development of improved subunit vaccines.     

Significance of composition and structure of the enterobacterial outer membrane to the transport of desired and undesired substances and its inhibition

The enterobacterial outer membrane forms a bilayer. Its outer monolyer consists of lipopolysaccharides and proteins, its inner monolayer of phospholipids and proteins. It thus represents an efficient penetration barrier against hydrophobic and anionic compounds (such as detergents or hydrophobic antibiotics) and against higher molecular substances (such as proteolytic, lipolytic, and murolylic enzymes). Some of the proteins (“porins”) form channels through the outer membrane through which neutral and cationic hydrophylic compounds up to a molecular weight of about 800 can pass. Besides the porins additional transport systems have been described. They play an important part in providing the bacteria with substances necessary for their growth, i.e., phosphate, iron ions, and others. Organic polycations are able to generate more or less severe disorganizations in the outer membrane through which they can pass the bilayer (“self-promoted pathway”). Some of these polycations represent efficient antibiotics (polymyxin B, nourseothricin). Bacteria are able to protect themselves against the harmful action of these substances by changing the composition of the outer membrane.     

Ion channel activities in the Escherichia coli outer membrane.

The electrical properties of Escherichia coli cells were examined by the patch-clamp technique. Giant cells or giant spheroplasts were generated by five different methods. By electron micrographic and other criteria we determined that the patches are most likely from the outer membrane. We regularly observed currents through at least two types of channels in this membrane. The first current is mechanosensitive and voltage-dependent, and can be observed in single gene mutants of the known major porins (ompF, ompC, phoE, lamB); this channel may represent a minor porin or a new class of outer membrane protein. The possible identity of the second, voltage-sensitive channel with one of the known outer membrane proteins is being explored. The high-resistance seals consistently formed on these patches and the presence of gated ion channels suggest that most of the pores of the outer membrane are not statically open, as commonly held, but are closed at rest and may be openable by physiological stimuli.     

High‐resolution architecture of the outer membrane of the Gram‐negative bacteria Roseobacter denitrificans

The outer membrane of Gram‐negative bacteria protects the cell against bactericidal substances. Passage of nutrients and waste is assured by outer membrane porins, beta‐barrel transmembrane channels. While atomic structures of several porins have been solved, so far little is known on the supramolecular structure of the outer membrane. Here we present the first high‐resolution view of a bacterial outer membrane gently purified maintaining remnants of peptidoglycan on the perisplasmic surface. Atomic force microscope images of outer membrane fragments of the size of ～50% of the bacterial envelope revealed that outer membrane porins are by far more densely packed than previously assumed. Indeed the outer membrane is a molecular sieve rather than a membrane. Porins cover ～70% of the membrane surface and form locally regular lattices. The potential role of exposed aromatic residues in the formation of the supramolecular assembly is discussed. Finally, we present first structural data of the outer membrane porin from the marine Gram‐negative bacteria Roseobacter denitrificans, and we perform a sequence alignment with porins of known structure.     

Molecular dynamics simulations of membrane proteins

Membrane proteins control the traffic across cell membranes and thereby play an essential role in cell function from transport of various solutes to immune response via molecular recognition. Because it is very difficult to determine the structures of membrane proteins experimentally, computational methods have been increasingly used to study their structure and function. Here we focus on two classes of membrane proteins—ion channels and transporters—which are responsible for the generation of action potentials in nerves, muscles, and other excitable cells. We describe how computational methods have been used to construct models for these proteins and to study the transport mechanism. The main computational tool is the molecular dynamics (MD) simulation, which can be used for everything from refinement of protein structures to free energy calculations of transport processes. We illustrate with specific examples from gramicidin and potassium channels and aspartate transporters how the function of these membrane proteins can be investigated using MD simulations.     

Membrane-derived oligosaccharides (MDO's) promote closing of an E. coli porin channel.

The outer membrane of Escherichia coli is a diffusion barrier for macromolecules, but allows the passage of small hydrophilic solutes through non-specific channels, the porins. Some electrophysiological studies find reconstituted porins in a mostly open state, while those done with the patch-clamp technique performed on live cells suggest that the vast majority of the native channels are closed. We present here current measurements through porins from reconstituted outer membrane, which demonstrate that bacterial metabolites, the MDO's, which bathe the periplasmic side of the outer membrane, induce the channels to close. These findings illustrate that the degree of openness of porins can be regulated by compounds naturally found in bacteria.     

Gating and conduction of nano-channel forming proteins: a computational approach

Monitoring conformational changes in ion channels is essential to understand their gating mechanism. Here, we explore the structural dynamics of four outer membrane proteins with different structures and functions in the slowest nonzero modes of vibration. Normal mode analysis was performed on the modified elastic network model of channel in the membrane. According to our results, when membrane proteins were analyzed in the dominant mode, the composed pores, TolC and α-hemolysin showed large motions at the intramembrane β-barrel region while, in other porins, OmpA and OmpF, largest motions observed in the region of external flexible loops. A criterion based on equipartition theorem was used to measure the possible amplitude of vibration in channel forming proteins. The current approach complements theoretical and experimental techniques including HOLE, Molecular Dynamics (MD), and voltage clamp used to address the channel’s structure and dynamics and provides the means to conduct a theoretical simultaneous study of the structure and function of the channel.

An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:3     

Transport across the bacterial outer membrane

Diffusion of small molecules across the outer membrane of gram-negative bacteria may occur through protein channels and through lipid bilayer domains. Among protein channels, many examples of trimeric porins, which produce water-filled diffusion channels, are known. Although the channels are nonspecific, the diffusion rates of solutes are often drastically affected by their gross physicochemical properties, such as size, charge, or lipophilicity, because the channel has a dimension not too different from that of the diffusing solutes. In the last few years, the structures of three such porins have been solved by X-ray crystallography. It is now known that a monomer unit traverses the membrane 16 times as -strands, and one of the external loop folds back into the channel to produce a narrow constriction. Most of the static properties of the channel, such as the pore size and the position of the amino acids that produce the constriction, can now be explained by the three-dimensional structure. Controversy, however, still surrounds the issue of whether there are dynamic modulation of the channel properties in response to pH, ionic strength, or membrane potential, and of whether such responses are physiological. More recently, two examples of monomeric porins have been identified. These porins allow a very slow diffusion of solutes, but the reason for this low permeability is still unclear. Finally, channels with specific binding sites facilitate the diffusion of specific classes of nutrients, often those compounds that are too large to penetrate rapidly through the porin channels. Lipid bilayers in the outer membrane were shown to be perhaps 50- to 100-fold less permeable to uncharged, lipophilic molecules in comparison with the bilayers made of the usual glycerophospholipids. This is caused by the presence of a lipopolysaccharide leaflet in the bilayer, and more specifically, by the presence of a larger number of fatty acids in each lipid molecule, and by the absence of unsaturated fatty acids in the lipopolysaccharide structure.     

Solute channels of the outer membrane: from bacteria to chloroplasts

Chloroplasts, unique organelles of plants, originated from endosymbiosis of an ancestor of today's cyanobacteria with a mitochondria-containing host cell. It is assumed that the outer envelope membrane, which delimits the chloroplast from the surrounding cytosol, was thus inherited from its Gram-negative bacterial ancestor. This plastid-specific membrane is thus equipped with elements of prokaryotic and eukaryotic origin. In particular, the membrane-intrinsic outer envelope proteins (OEPs) form solute channels with properties reminiscent of porins and channels in the bacterial outer membrane. OEP channels are characterised by distinct specificities for metabolites and a quite peculiar expression pattern in specialised plant organs and plastids, thus disproving the assumption that the outer envelope is a non-specific molecular sieve. The same is true for the outer membrane of Gram-negative bacteria, which functions as a permeability barrier in addition to the cytoplasmic membrane, and embeds different classes of channel pores. The channels of these prokaryotic prototype proteins, ranging from unspecific porins to specific channels to ligand-gated receptors, are exclusively built of beta-barrels. Although most of the OEP channels are formed by beta-strands as well, phylogeny based on sequence homology alone is not feasible. Thus, the comparison of structural and functional properties of chloroplast outer envelope and bacterial outer membrane channels is required to pinpoint the ancestral OEP 'portrait gallery'.     

Structure and function of pore-forming proteins from bacteria of the genus Yersinia: I. Isolation and a comparison of physicochemical properties and functional activity of Yersinia porins

The molecular organization and functional activity of porins isolated from the outer membrane (OM) of the Yersinia enterocolitica and three phylogenetically close nonpathogenic Yersinia species (Y. intermedia, Y. kristensenii, and Y. frederiksenii) cultured at 6-8 degrees C were comparatively studied for the first time. The proteins were isolated in two molecular forms (trimeric and monomeric), and their spatial structures were characterized by the methods of optical spectroscopy, CD and intrinsic protein fluorescence. The studied porins were shown to belong to the beta-structural proteins (they have 59-96% total beta structures and 0-17% alpha helices). The spatial structures of the proteins were demonstrated to depend on the nature of the detergent used for solubilization. Unlike the enterobacterial pore-forming proteins, the porin trimers are less stable to sodium dodecyl sulfate (SDS). The spatial structures of the porins become more compact after the substitution of octyl beta-D-glucoside for SDS: the content of beta structures increases and the accessibility of Trp residues to solvent decreases. It was established with the use of the technique of bilayer lipid membranes that the functional properties of the porins are similar to those of the OmpF proteins of Gram-negative bacteria. Trimers are functionally active forms of the porins. Special features of the pore-forming activity of the Yersinia porins were revealed to depend on the microorganism species and the value of the membrane potential.     

Molecular dynamics simulations of proteins in lipid bilayers

With recent advances in X-ray crystallography of membrane proteins promising many new high-resolution structures, molecular dynamics simulations will become increasingly valuable for understanding membrane protein function, as they can reveal the dynamic behavior concealed in the static structures. Dramatic increases in computational power, in synergy with more efficient computational methodologies, now allow us to carry out molecular dynamics simulations of any structurally known membrane protein in its native environment, covering timescales of up to 0.1 micros. At the frontiers of membrane protein simulations are ion channels, aquaporins, passive and active transporters, and bioenergetic proteins.     

A 3D model of the voltage-dependent anion channel (VDAC)

Eukaryotic porins are a group of membrane proteins whose best known role is to form an aqueous pore channel in the mitochondrial outer membrane. As opposed to the bacterial porins (a large family of protein whose 3D structure has been determined by X-ray diffraction), the structure of eukaryotic porins (also termed VDACs, voltage-dependent anion-selective channels) is still a matter of debate. We analysed the secondary structure of VDAC from the yeast Saccharomyces cerevisiae, the fungus Neurospora crassa and the mouse with different types of neural network-based predictors. The predictors were able to discriminate membrane β-strands, globular -helices and membrane -helices and localised, in all three VDAC sequences, 16 β-strands along the chain. For all three sequences the N-terminal region showed a high propensity to form a globular -helix. The 16 β-strand VDAC motif was thus aligned to a bacterial porin-derived template containing a similar 16 β-strand motif. The alignment of the VDAC sequence with the bacterial porin sequence was used to compute a set of 3D coordinates, which constitutes the first 3D prediction of a eukaryotic porin. All the predicted structures assume a β-barrel structure composed of 16 β-strands with the N-terminus outside the membrane. Loops are shorter in this side of the membrane than in the other, where two long loops are protruding. The shape of the pore varies between almost circular for Neurospora and mouse and slightly oval for yeast. Average values between 3 and 2.5 nm at the C-carbon backbone are found for the diameter of the channels. In this model VDAC shows large portions of the structure exposed on both sides of the membrane. The architecture we determine allows speculation about the mechanism of possible interactions between VDAC and other proteins on both sides of the mitochondrial outer membrane. The computed 3D model is consistent with most of the experimental results so far reported.     

Understanding Voltage Gating of Providencia stuartii Porins at Atomic Level

Bacterial porins are water-filled β-barrel channels that allow translocation of solutes across the outer membrane. They feature a constriction zone, contributed by the plunging of extracellular loop 3 (L3) into the channel lumen. Porins are generally in the open state, but undergo gating in response to external voltages. To date the underlying mechanism is unclear. Here we report results from molecular dynamics simulations on the two porins of Providenica stuartii, Omp-Pst1 and Omp-Pst2, which display distinct voltage sensitivities. Voltage gating was observed in Omp-Pst2, where the binding of cations in-between L3 and the barrel wall results in exposing a conserved aromatic residue in the channel lumen, thereby halting ion permeation. Comparison of Omp-Pst1 and Omp-Pst2 structures and trajectories suggests that their sensitivity to voltage is encoded in the hydrogen-bonding network anchoring L3 onto the barrel wall, as we observed that it is the strength of this network that governs the probability of cations binding behind L3. That Omp-Pst2 gating is observed only when ions flow against the electrostatic potential gradient of the channel furthermore suggests a possible role for this porin in the regulation of charge distribution across the outer membrane and bacterial homeostasis.     

Structure and function of bacterial outer membrane proteins: barrels in a nutshell

The outer membrane protects Gram-negative bacteria against a harsh environment. At the same time, the embedded proteins fulfil a number of tasks that are crucial to the bacterial cell, such as solute and protein translocation, as well as signal transduction. Unlike membrane proteins from all other sources, integral outer membrane proteins do not consist of transmembrane alpha-helices, but instead fold into antiparallel beta-barrels. Over recent years, the atomic structures of several outer membrane proteins, belonging to six families, have been determined. They include the OmpA membrane domain, the OmpX protein, phospholipase A, general porins (OmpF, PhoE), substrate-specific porins (LamB, ScrY) and the TonB-dependent iron siderophore transporters FhuA and FepA. These crystallographic studies have yielded invaluable insight into and decisively advanced the understanding of the functions of these intriguing proteins. Our review is aimed at discussing their common principles and peculiarities as well as open questions associated with them.     

OmpA: Gating and dynamics via molecular dynamics simulations

Outer membrane proteins (OMPs) of Gram-negative bacteria have a variety of functions including passive transport, active transport, catalysis, pathogenesis and signal transduction. Whilst the structures of ∼ 25 OMPs are currently known, there is relatively little known about their dynamics in different environments. The outer membrane protein, OmpA from Escherichia coli has been studied extensively in different environments both experimentally and computationally, and thus provides an ideal test case for the study of the dynamics and environmental interactions of outer membrane proteins. We review molecular dynamics simulations of OmpA and its homologues in a variety of different environments and discuss possible mechanisms of pore gating. The transmembrane domain of E. coli OmpA shows subtle differences in dynamics and interactions between a detergent micelle and a lipid bilayer environment. Simulations of the crystallographic unit cell reveal a micelle-like network of detergent molecules interacting with the protein monomers. Simulation and modelling studies emphasise the role of an electrostatic-switch mechanism in the pore-gating mechanism. Simulation studies have been extended to comparative models of OmpA homologues from Pseudomonas aeruginosa (OprF) and Pasteurella multocida (PmOmpA), the latter model including the periplasmic C-terminal domain.     

Structure and function of pore-forming proteins from bacteria of the genus Yersinia: I. Isolation and a comparison of physicochemical properties and functional activity of Yersinia porins

The molecular organization and functional activity of porins isolated from the outer membrane (OM) of the Yersinia enterocolitica and three phylogenetically close nonpathogenic Yersinia species (Y. intermedia, Y. kristensenii, and Y. frederiksenii) cultured at 6–8°C were comparatively studied for the first time. The proteins were isolated in two molecular forms (trimeric and monomeric), and their spatial structures were characterized by the methods of optical spectroscopy, CD and intrinsic protein fluorescence. The studied porins were shown to belong to the β-structural proteins (they have 59–96% total β structures and 0–17% α helices). The spatial structures of the proteins were demonstrated to depend on the nature of the detergent used for solubilization. Unlike the enterobacterial pore-forming proteins, the porin trimers are less stable to sodium dodecyl sulfate (SDS). The spatial structures of the porins become more compact after the substitution of octyl β-D-glucoside for SDS: the content of β structures increases and the accessibility of Trp residues to solvent decreases. It was established with the use of the technique of bilayer lipid membranes that the functional properties of the porins are similar to those of the OmpF proteins of Gram-negative bacteria. Trimers are functionally active forms of the porins. Special features of the pore-forming activity of the Yersinia porins were revealed to depend on the microorganism species and the value of the membrane potential.     

Membrane proteins: molecular dynamics simulations

Molecular dynamics simulations of membrane proteins are making rapid progress, because of new high-resolution structures, advances in computer hardware and atomistic simulation algorithms, and the recent introduction of coarse-grained models for membranes and proteins. In addition to several large ion channel simulations, recent studies have explored how individual amino acids interact with the bilayer or snorkel/anchor to the headgroup region, and it has been possible to calculate water/membrane partition free energies. This has resulted in a view of bilayers as being adaptive rather than purely hydrophobic solvents, with important implications, for example, for interaction between lipids and arginines in the charged S4 helix of voltage-gated ion channels. However, several studies indicate that the typical current simulations fall short of exhaustive sampling, and that even simple protein-membrane interactions require at least ca. 1mus to fully sample their dynamics. One new way this is being addressed is coarse-grained models that enable mesoscopic simulations on multi-mus scale. These have been used to model interactions, self-assembly and membrane perturbations induced by proteins. While they cannot replace all-atom simulations, they are a potentially useful technique for initial insertion, placement, and low-resolution refinement.     

Origami in the outer membrane: the transmembrane arrangement of mitochondrial porins.

Voltage-dependent anion-selective channels (VDAC), also known as mitochondrial porins, are key regulators of metabolite flow across the mitochondrial outer membrane. Porins from a wide variety of organisms share remarkably similar electrophysiological properties, in spite of considerable sequence dissimilarity, indicating that they share a common structure. Based on primary sequence considerations, analogy with bacterial porins, and circular dichroism analysis, it is agreed that VDAC spans the outer membrane as a beta-barrel. However, the residues that form the antiparallel beta-strands comprising this barrel remain unknown. Various predictive methods, largely based on the known structures of bacterial beta-barrels, have been applied to the primary sequences of VDAC. Refinement and confirmation of these predictions have developed through numerous investigations of wild-type and variant porins, both in mitochondria and in artificial membranes. These experiments have involved VDAC from several sources, precluding the generation of a unified model. Herein, using the Neurospora VDAC sequence as a template, the published structural information and predictions have been reassessed to delineate a model that satisfies most of the available data.