Cumulative hydropathic topology of a voltage-gated sodium channel at atomic resolution.

Voltage-gated sodium channels (NavChs) are biological pores that control the flow of sodium ions through the cell membrane. In humans, mutations in genes encoding NavChs can disrupt physiological cellular activity thus leading to a wide spectrum of diseases. Here, we present a topological connection between the functional architecture of a NavAb bacterial channel and accumulation of atomic hydropathicity around its pore. This connection is established via a scaling analysis methodology that elucidates how intrachannel hydropathic density variations translate into hydropathic dipole field configurations along the pore. Our findings suggest the existence of a nonrandom cumulative hydropathic topology that is organized parallel to the membrane surface so that pore's stability, as well as, gating behavior are guaranteed. Given the biophysical significance of the hydropathic effect, our study seeks to provide a computational framework for studying cumulative hydropathic topological properties of NavChs and pore-forming proteins in general.

Nanodisc technology facilitates identification of monoclonal antibodies targeting multi-pass membrane proteins.

Multi-pass membrane proteins are important targets of biologic medicines. Given the inherent difficulties in working with membrane proteins, we sought to investigate the utility of membrane scaffold protein nanodiscs as a means of solubilizing membrane proteins to aid antibody discovery. Using a model multi-pass membrane protein, we demonstrate how incorporation of a multi-pass membrane protein into nanodiscs can be used in flow cytometry to identify antigen-specific hybridoma. The use of target protein-loaded nanodiscs to sort individual hybridoma early in the screening process can reduce the time required to identify antibodies against multi-pass membrane proteins.

Structural basis for efonidipine block of a voltage-gated Ca2+ channel.

Efonidipine is a dual L-/T- type calcium channel blocker with a slow onset of action and a long lasting effect that exibihits antihypertensive and nephroprotective effects. differs from most other DHPs which can induce reflex tachycardia. Efonidipine reduces blood pressure without decreasing cardiac output and exerts organ-protective effects on the heart and kidney. In order to investigate how efonidipine block voltage-gated Ca2+ channel, we determined the crystal structure of CaVAb in complex with efonidipine at atomic resolution using x-ray crystallography. Our results reveal that efonidipine targets the central cavity of a model voltage-gated calcium channel underneath its selectivity filter and occlude the channel in an inactivated state. Binding of efonidipine does not break down the fourfold symmetry of the quaternary structure and its pore structure. Our work provides the structural basis for efonidipine block of a voltage-gated Ca2+ channel at the molecular level.

Simulation of the effect of an external GHz electric field on the potential energy profile of Ca2+ ions in the selectivity filter of the CaV Ab channel.

CaV channels are transmembrane proteins that mediate and regulate ion fluxes across cell membranes, and they are activated in response to action potentials to allow Ca2+ influx. Since ion channels are composed of charge or polar groups, an external alternating electric field may affect the ion-selective membrane transport and the performance of the channel. In this article, we have investigated the effect of an external GHz electric field on the dynamics of calcium ions in the selectivity filter of the CaV Ab channel. Molecular dynamics (MD) simulations and the potential of mean force (PMF) calculations were carried out, via the umbrella sampling method, to determine the free energy profile of Ca2+ ions in the CaV Ab channels in presence and absence of an external field. Exposing CaV Ab channel to 1, 2, 3, 4, and 5 GHz electric fields increases the depth of the potential energy well and this may result in an increase in the affinity and strength of Ca2+ ions to binding sites in the selectivity filter the channel. This increase of strength of Ca2+ ions binding in the selectivity filter may interrupt the mechanism of Ca2+ ion conduction, and leads to a reduction of Ca2+ ion permeation through the CaV Ab channel.

Arcobacter acticola sp. nov., isolated from seawater on the East Sea in South Korea.

A Gram-stain-negative, facultative aerobic, non-flagellated, and rod-shaped bacterium, designated AR-13(T), was isolated from a seawater on the East Sea in South Korea, and subjected to a polyphasic taxonomic study. Strain AR-13(T) grew optimally at 30°C, at pH 7.0-8.0 and in the presence of 0-0.5% (w/v) NaCl. The phylogenetic trees based on 16S rRNA gene sequences showed that strain AR-13(T) fell within the clade comprising the type strains of Arcobacter species, clustering coherently with the type strain of Arcobacter venerupis. Strain AR-13(T) exhibited 16S rRNA gene sequence similarity values of 98.1% to the type strain of A. venerupis and of 93.2-96.9% to the type strains of the other Arcobacter species. Strain AR-13(T) contained MK-6 as the only menaquinone and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c), C16:0, C18:1 ω7c, and summed feature 2 (iso-C16:1 I and/or C14:0 3-OH) as the major fatty acids. The major polar lipids detected in strain AR-13(T) were phosphatidylethanolamine, phosphatidylglycerol, and one unidentified aminophospholipid. The DNA G+C content was 28.3 mol% and its mean DNA-DNA relatedness value with the type strain of A. venerupis was 21%. Differential phenotypic properties, together with its phylogenetic and genetic distinctiveness, revealed that strain AR-13(T) is separated from recognized Arcobacter species. On the basis of the data presented, strain AR-13(T) is considered to represent a novel species of the genus Arcobacter, for which the name Arcobacter acticola sp. nov. is proposed. The type strain is AR-13(T) (=KCTC 52212(T) =NBRC 112272(T)).

Ion conduction and conformational flexibility of a bacterial voltage-gated sodium channel.

Voltage-gated Na(+) channels play an essential role in electrical signaling in the nervous system and are key pharmacological targets for a range of disorders. The recent solution of X-ray structures for the bacterial channel NavAb has provided an opportunity to study functional mechanisms at the atomic level. This channel's selectivity filter exhibits an EEEE ring sequence, characteristic of mammalian Ca(2+), not Na(+), channels. This raises the fundamentally important question: just what makes a Na(+) channel conduct Na(+) ions? Here we explore ion permeation on multimicrosecond timescales using the purpose-built Anton supercomputer. We isolate the likely protonation states of the EEEE ring and observe a striking flexibility of the filter that demonstrates the necessity for extended simulations to study conduction in this channel. We construct free energy maps to reveal complex multi-ion conduction via knock-on and "pass-by" mechanisms, involving concerted ion and glutamate side chain movements. Simulations in mixed ionic solutions reveal relative energetics for Na(+), K(+), and Ca(2+) within the pore that are consistent with the modest selectivity seen experimentally. We have observed conformational changes in the pore domain leading to asymmetrical collapses of the activation gate, similar to proposed inactivated structures of NavAb, with helix bending involving conserved residues that are critical for slow inactivation. These structural changes are shown to regulate access to fenestrations suggested to be pathways for lipophilic drugs and provide deeper insight into the molecular mechanisms connecting drug activity and slow inactivation.

Fourier transform infrared spectroscopy as a novel approach for analyzing the biochemical effects of anionic surfactants on a surfactant-degrading Arcobacter butzleri strain.

Anionic surfactant-biodegrading capability of an Arcobacter butzleri strain was analyzed under aerobic conditions. The A. butzleri isolate displayed efficient surfactant-biodegrading capacity for sodium dodecyl sulfate (SDS) at concentrations of up to 100 mg/L in 6 days, corresponding to 99.0% removal efficiency. Fourier transform infrared spectroscopy was applied to observe the effects of varying concentrations of SDS on the biochemistry of bacterial cells. Results suggest that protein secondary structures were altered in bacterial cells at sufficiently high SDS concentrations, concurrent with SDS biodegradation.

Arcobacter cloacae sp. nov. and Arcobacter suis sp. nov., two new species isolated from food and sewage.

Three strains recovered from mussels (F26), sewage (SW28-13(T)) and pork meat (F41(T)) were characterized as Arcobacter. They did not appear to resemble any known species on the basis of their 16S rDNA-RFLP patterns and the rpoB gene analyses. However, strains F26 and SW28-13(T) appeared to be the same species. The 16S rRNA gene sequence similarity of strains SW28-13(T) and F41(T) to the type strains of all other Arcobacter species ranged from 94.1% to 99.6% and 93.4% to 98.8%, respectively. Phenotypic characteristics and the DNA-DNA hybridization (DDH) results showed that they belonged to 2 new Arcobacter species. A multilocus phylogenetic analysis (MLPA) with the concatenated sequences of 5 housekeeping genes (gyrA, atpA, rpoB, gyrB and hsp60) was used for the first time in the genus, showing concordance with the 16S rRNA gene phylogenetic analysis and DDH results. The MALDI-TOF mass spectra also discriminated these strains as two new species. The names proposed for them are Arcobacter cloacae with the type strain SW28-13(T) (=CECT 7834(T)=LMG 26153(T)) and Arcobacter suis with the type strain F41(T) (=CECT 7833(T)=LMG 26152(T)).

Crystal structure of a voltage-gated sodium channel in two potentially inactivated states.

In excitable cells, voltage-gated sodium (Na(V)) channels activate to initiate action potentials and then undergo fast and slow inactivation processes that terminate their ionic conductance. Inactivation is a hallmark of Na(V) channel function and is critical for control of membrane excitability, but the structural basis for this process has remained elusive. Here we report crystallographic snapshots of the wild-type Na(V)Ab channel from Arcobacter butzleri captured in two potentially inactivated states at 3.2 Å resolution. Compared to previous structures of Na(V)Ab channels with cysteine mutations in the pore-lining S6 helices (ref. 4), the S6 helices and the intracellular activation gate have undergone significant rearrangements: one pair of S6 helices has collapsed towards the central pore axis and the other S6 pair has moved outward to produce a striking dimer-of-dimers configuration. An increase in global structural asymmetry is observed throughout our wild-type Na(V)Ab models, reshaping the ion selectivity filter at the extracellular end of the pore, the central cavity and its residues that are analogous to the mammalian drug receptor site, and the lateral pore fenestrations. The voltage-sensing domains have also shifted around the perimeter of the pore module in wild-type Na(V)Ab, compared to the mutant channel, and local structural changes identify a conserved interaction network that connects distant molecular determinants involved in Na(V) channel gating and inactivation. These potential inactivated-state structures provide new insights into Na(V) channel gating and novel avenues to drug development and therapy for a range of debilitating Na(V) channelopathies.

Arcobacter bivalviorum sp. nov. and Arcobacter venerupis sp. nov., new species isolated from shellfish.

A group of ten Arcobacter isolates (Gram negative, slightly curved motile rods, oxidase positive) was recovered from mussels (nine) and from clams (one). These isolates could not be assigned to any known species using the molecular identification methods specific for this genus (16S rDNA-RFLP and m-PCR). The aim of this study is to establish the taxonomic position of these isolates. The 16S rRNA gene sequence similarity of mussel strain F4(T) to the type strains of all other Arcobacter species ranged from 91.1% to 94.8%. The species most similar to the clams' strain F67-11(T) were Arcobacter defluvii (CECT 7697(T), 97.1%) and Arcobacter ellisii (CECT 7837(T), 97.0%). On the basis of phylogenetic analyses with 16S rRNA, rpoB, gyrB and hsp60 genes, the mussel and clam strains formed two different, new lineages within the genus Arcobacter. These data, together with their different phenotypic characteristics and MALDI-TOF mass spectra, revealed that these strains represent two new species, for which the names Arcobacter bivalviorum (type strain F4(T)=CECT 7835(T)=LMG 26154(T)) and Arcobacter venerupis (type strain F67-11(T)=CECT 7836(T)=LMG 26156(T)) are proposed.

Mechanism of ion permeation and selectivity in a voltage gated sodium channel.

The rapid and selective transport of Na(+) through sodium channels is essential for initiating action potentials within excitable cells. However, an understanding of how these channels discriminate between different ion types and how ions permeate the pore has remained elusive. Using the recently published crystal structure of a prokaryotic sodium channel from Arcobacter butzleri, we are able to determine the steps involved in ion transport and to pinpoint the location and likely mechanism used to discriminate between Na(+) and K(+). Na(+) conduction is shown to involve the loosely coupled "knock-on" movement of two solvated ions. Selectivity arises due to the inability of K(+) to fit between a plane of glutamate residues with the preferred solvation geometry that involves water molecules bridging between the ion and carboxylate groups. These mechanisms are different to those described for K(+) channels, highlighting the importance of developing a separate mechanistic understanding of Na(+) and Ca(2+) channels.

The crystal structure of a voltage-gated sodium channel.

Voltage-gated sodium (Na(V)) channels initiate electrical signalling in excitable cells and are the molecular targets for drugs and disease mutations, but the structural basis for their voltage-dependent activation, ion selectivity and drug block is unknown. Here we report the crystal structure of a voltage-gated Na(+) channel from Arcobacter butzleri (NavAb) captured in a closed-pore conformation with four activated voltage sensors at 2.7 Šresolution. The arginine gating charges make multiple hydrophilic interactions within the voltage sensor, including unanticipated hydrogen bonds to the protein backbone. Comparisons to previous open-pore potassium channel structures indicate that the voltage-sensor domains and the S4-S5 linkers dilate the central pore by pivoting together around a hinge at the base of the pore module. The NavAb selectivity filter is short, ∼4.6 Šwide, and water filled, with four acidic side chains surrounding the narrowest part of the ion conduction pathway. This unique structure presents a high-field-strength anionic coordination site, which confers Na(+) selectivity through partial dehydration via direct interaction with glutamate side chains. Fenestrations in the sides of the pore module are unexpectedly penetrated by fatty acyl chains that extend into the central cavity, and these portals are large enough for the entry of small, hydrophobic pore-blocking drugs. This structure provides the template for understanding electrical signalling in excitable cells and the actions of drugs used for pain, epilepsy and cardiac arrhythmia at the atomic level.

The structure of the carbohydrate backbone of the lipooligosaccharide from the halophilic bacterium Arcobacter halophilus.

A novel oligosaccharide was isolated and identified from the lipooligosaccharide fraction of the halophilic marine bacterium Arcobacter halophilus. The complete structure was achieved by chemical analysis, 2D NMR spectroscopy, and MALDI mass spectrometry as the following: alpha-Glc-(1-->7)-alpha-Hep-(1-->5)-alpha-Kdo4P-(2-->6)-beta-GlcN4P-(1-->6)-alpha-GlcN1P.

Susceptibility of Arcobacter butzleri to human blood serum / Susceptibilidade de Arcobacter butzleri ao soro humano

A susceptibilidade de 10 amostras de Arcobacter butzleri ao soro humano foi estudada. A maior atividade bactericida foi encontrada no soro humano normal, com taxas de sobrevivência bacterianainversamente proporcionais à diluição do soro. As maiores taxas de sobrevivência foram obtidas com o soro inativado pelo calor. As taxas de sobrevivência decresceram com a adição de soro fresco ao inativado. O soro com valores reduzidos de gamaglobulinas e valores normais de complemento mostrou ativo efeito bactericida. Os resultados demonstraram que A. butzleri é altamente susceptível ao efeito bactericida do soro humano, sugerindo que pode ser capaz de ativar diretamente o complemento pela via alternativa.

Species-specific identification and differentiation of Arcobacter, Helicobacter and Campylobacter by full-spectral matrix-associated laser desorption/ionization time of flight mass spectrometry analysis.

Rapid and reliable identification of Arcobacter and Helicobacter species, and their distinction from phenotypically similar Campylobacter species, has become increasingly important, since many of them are now recognized as human and/or animal pathogens. Matrix-associated laser desorption/ionization-time of flight (MALDI-TOF) MS has been shown to be a rapid and sensitive method for characterization of micro-organisms. In this study, we therefore established a reference database of selected Arcobacter, Helicobacter and Campylobacter species for MALDI-TOF MS identification. Besides the species with significance as food-borne pathogens - Arcobacter butzleri, Helicobacter pullorum, Campylobacter jejuni and Campylobacter coli - several other members of these genera were included in the reference library to determine the species specificity of the designed MALDI Biotyper reference database library. Strains that made up the reference database library were grown on Columbia agar, and yielded reproducible and unique mass spectra profiles, which were compared with the Bruker Biotyper database, version 2. The database was used to identify 144 clinical isolates using whole spectral profiles. Furthermore, reproducibility of MALDI-TOF MS results was evaluated with respect to age and/or storage of bacteria and different growth media. It was found that correct identification could be obtained even if the bacteria were stored at room temperature or at 4 degrees C up to 9 days before being tested. In addition, bacteria were correctly identified when grown on Campylosel agar; however, they were not when grown on modified charcoal cefoperazone deoxycholate agar. These results indicate that MALDI-TOF MS fingerprinting is a fast and reliable method for the identification of Arcobacter and Helicobacter species, and their distinction from phenotypically similar Campylobacter species, with applications in clinical diagnostics.

Characterization of an autotrophic sulfide-oxidizing marine Arcobacter sp. that produces filamentous sulfur.

A coastal marine sulfide-oxidizing autotrophic bacterium produces hydrophilic filamentous sulfur as a novel metabolic end product. Phylogenetic analysis placed the organism in the genus Arcobacter in the epsilon subdivision of the Proteobacteria. This motile vibrioid organism can be considered difficult to grow, preferring to grow under microaerophilic conditions in flowing systems in which a sulfide-oxygen gradient has been established. Purified cell cultures were maintained by using this approach. Essentially all 4',6-diamidino-2-phenylindole dihydrochloride-stained cells in a flowing reactor system hybridized with Arcobacter-specific probes as well as with a probe specific for the sequence obtained from reactor-grown cells. The proposed provisional name for the coastal isolate is "Candidatus Arcobacter sulfidicus." For cells cultured in a flowing reactor system, the sulfide optimum was higher than and the CO(2) fixation activity was as high as or higher than those reported for other sulfur oxidizers, such as Thiomicrospira spp. Cells associated with filamentous sulfur material demonstrated nitrogen fixation capability. No ribulose 1,5-bisphosphate carboxylase/oxygenase could be detected on the basis of radioisotopic activity or by Western blotting techniques, suggesting an alternative pathway of CO(2) fixation. The process of microbial filamentous sulfur formation has been documented in a number of marine environments where both sulfide and oxygen are available. Filamentous sulfur formation by "Candidatus Arcobacter sulfidicus" or similar strains may be an ecologically important process, contributing significantly to primary production in such environments.