Gloeobacter Research Articles

Poles Apart: Arctic and Antarctic Octadecabacter strains Share High Genome Plasticity and a New Type of Xanthorhodopsin

The genus Octadecabacter is a member of the ubiquitous marine Roseobacter clade. The two described species of this genus, Octadecabacter arcticus and Octadecabacter antarcticus, are psychrophilic and display a bipolar distribution. Here we provide the manually annotated and finished genome sequences of the type strains O. arcticus 238 and O. antarcticus 307, isolated from sea ice of the Arctic and Antarctic, respectively. Both genomes exhibit a high genome plasticity caused by an unusually high density and diversity of transposable elements. This could explain the discrepancy between the low genome synteny and high 16S rRNA gene sequence similarity between both strains. Numerous characteristic features were identified in the Octadecabacter genomes, which show indications of horizontal gene transfer and may represent specific adaptations to the habitats of the strains. These include a gene cluster encoding the synthesis and degradation of cyanophycin in O. arcticus 238, which is absent in O. antarcticus 307 and unique among the Roseobacter clade. Furthermore, genes representing a new subgroup of xanthorhodopsins as an adaptation to icy environments are present in both Octadecabacter strains. This new xanthorhodopsin subgroup differs from the previously characterized xanthorhodopsins of Salinibacter ruber and Gloeobacter violaceus in phylogeny, biogeography and the potential to bind 4-keto-carotenoids. Biochemical characterization of the Octadecabacter xanthorhodopsins revealed that they function as light-driven proton pumps.

Experimental determination of the vertical alignment between the second and third transmembrane segments of muscle nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChR) are members of the Cys-loop ligand-gated ion channel superfamily. Muscle nAChR are heteropentamers that assemble from two α, and one each of β, γ, and δ subunits. Each subunit is composed of three domains, extracellular, transmembrane and intracellular. The transmembrane domain consists of four α-helical segments (M1-M4). Pioneering structural information was obtained using electronmicroscopy of Torpedo nAChR. The recently solved X-ray structure of the first eukaryotic Cys-loop receptor, a truncated (intracellular domain missing) glutamate-gated chloride channel α (GluClα) showed the same overall architecture. However, a significant difference with regard to the vertical alignment between the channel-lining segment M2 and segment M3 was observed. Here, we used functional studies utilizing disulfide trapping experiments in muscle nAChR to determine the spatial orientation between M2 and M3. Our results are in agreement with the vertical alignment as obtained when using the GluClα structure as a template to homology model muscle nAChR, however, they cannot be reconciled with the current Torpedo nAChR model. The vertical M2-M3 alignments as observed in X-ray structures of prokaryotic Gloeobacter violaceus ligand-gated ion channel and GluClα are in agreement. Our results further confirm that this alignment in Cys-loop receptors is conserved between prokaryotes and eukaryotes.

Structural basis for potentiation by alcohols and anaesthetics in a ligand-gated ion channel

Ethanol alters nerve signalling by interacting with proteins in the central nervous system, particularly pentameric ligand-gated ion channels. A recent series of mutagenesis experiments on Gloeobacter violaceus ligand-gated ion channel, a prokaryotic member of this family, identified a single-site variant that is potentiated by pharmacologically relevant concentrations of ethanol. Here we determine crystal structures of the ethanol-sensitized variant in the absence and presence of ethanol and related modulators, which bind in a transmembrane cavity between channel subunits and may stabilize the open form of the channel. Structural and mutagenesis studies defined overlapping mechanisms of potentiation by alcohols and anaesthetics via the inter-subunit cavity. Furthermore, homology modelling show this cavity to be conserved in human ethanol-sensitive glycine and GABA(A) receptors, and to involve residues previously shown to influence alcohol and anaesthetic action on these proteins. These results suggest a common structural basis for ethanol potentiation of an important class of targets for neurological actions of ethanol.

Multisite Binding of a General Anesthetic to the Prokaryotic Pentameric Erwinia chrysanthemi Ligand-gated Ion Channel (ELIC)

Pentameric ligand-gated ion channels (pLGICs), such as nicotinic acetylcholine, glycine, γ-aminobutyric acid GABA(A/C) receptors, and the Gloeobacter violaceus ligand-gated ion channel (GLIC), are receptors that contain multiple allosteric binding sites for a variety of therapeutics, including general anesthetics. Here, we report the x-ray crystal structure of the Erwinia chrysanthemi ligand-gated ion channel (ELIC) in complex with a derivative of chloroform, which reveals important features of anesthetic recognition, involving multiple binding at three different sites. One site is located in the channel pore and equates with a noncompetitive inhibitor site found in many pLGICs. A second transmembrane site is novel and is located in the lower part of the transmembrane domain, at an interface formed between adjacent subunits. A third site is also novel and is located in the extracellular domain in a hydrophobic pocket between the β7-β10 strands. Together, these results extend our understanding of pLGIC modulation and reveal several specific binding interactions that may contribute to modulator recognition, further substantiating a multisite model of allosteric modulation in this family of ion channels.

Multiscale modeling of solvation in chemical and biological nanosystems and in nanoporous materials

Statistical–mechanical, 3D-RISM-KH molecular theory of solvation (3D reference interaction site model with the Kovalenko–Hirata closure) is promising as an essential part of multiscale methodology for chemical and biomolecular nanosystems in solution. 3D-RISM-KH explains the molecular mechanisms of self-assembly and conformational stability of synthetic organic rosette nanotubes (RNTs), aggregation of prion proteins and β-sheet amyloid oligomers, protein-ligand binding, and function-related solvation properties of complexes as large as the Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC) and GroEL/ES chaperone. Molecular mechanics/Poisson–Boltzmann (generalized Born) surface area [MM/PB(GB)SA] post-processing of molecular dynamics (MD) trajectories involving SA empirical nonpolar terms is replaced with MM/3D-RISM-KH statistical–mechanical evaluation of the solvation thermodynamics. 3D-RISM-KH has been coupled with multiple time-step (MTS) MD of the solute biomolecule driven by effective solvation forces, which are obtained analytically by converging the 3D-RISM-KH integral equations at outer time-steps and are calculated in between by using solvation force coordinate extrapolation (SFCE) in the subspace of previous solutions to 3D-RISM-KH. The procedure is stabilized by the optimized isokinetic Nosé–Hoover (OIN) chain thermostatting, which enables gigantic outer time-steps up to picoseconds to accurately calculate equilibrium properties. The multiscale OIN/SFCE/3D-RISM-KH algorithm is implemented in the Amber package and illustrated on a fully flexible model of alanine dipeptide in aqueous solution, exhibiting the computational rate of solvent sampling 20 times faster than standard MD with explicit solvent. Further substantial acceleration can be achieved with 3D-RISM-KH efficiently sampling essential events with rare statistics such as exchange and localization of solvent, ions, and ligands at binding sites and pockets of the biomolecule. 3D-RISM-KH was coupled with ab initio complete active space self-consistent field (CASSCF) and orbital-free embedding (OFE) Kohn–Sham (KS) density functional theory (DFT) quantum chemistry methods in an SCF description of electronic structure, optimized geometry, and chemical reactions in solution. The (OFE)KS-DFT/3D-RISM-KH multi-scale method is implemented in the Amsterdam Density Functional (ADF) package and extensively validated against experiment for solvation thermochemistry, photochemistry, conformational equilibria, and activation barriers of various nanosystems in solvents and ionic liquids (ILs). Finally, the replica RISM-KH-VM molecular theory for the solvation structure, thermodynamics, and electrochemistry of electrolyte solutions sorbed in nanoporous materials reveals the molecular mechanisms of sorption and supercapacitance in nanoporous carbon electrodes, which is drastically different from a planar electrical double layer.

Study of the Interaction between General Anesthetics and a Bacterial Homologue to the Human Nicotinic Receptor

Pentameric Ligand Gated Ion Channels (pLGICs) are membrane receptors widely spread in the animal kingdom that play a key role in the nervous signal transduction. The discovery and the crystallization of bacterial homologues, such as GLIC from Gloeobacter violaceus in 2008 (Bocquet et al., Nature, 2008, 457:111; Hilf & Dutzler, Nature, 2008, 457:115) and of a novel locally-closed form (Nury et al. Nature, 2011, 469:428) provided new insights in the understanding of the operating mechanism of these channels. General anesthetics (GAs) such as propofol and desflurane could be co-crystallized with GLIC (see for example Nury et al., Nature, 2011, 469:428). The potential existence of several binding sites for GAs, alcohols and ions that modulate the activity of the channel is currently debated. Here, we intend to study the dynamic properties of the interactions between GLIC-desflurane and GLIC-propofol by means of molecular dynamics simulations, on the basis of existing crystal structures of open and locally-closed forms of the channel. We computed more than one hundred all-atom simulations of the ligand-bound GLIC system inserted in a membrane to obtain significant statistics on the exploration of the cavity by desflurane and propofol. We complete this study by free energy calculations to estimate the affinity of propofol and desflurane to both the inter- and intra-subunit cavities. This approach may allow us to explore the ligand-filled cavity rather exhaustively and provide a sound background for deriving hypotheses on GAs.

Interaction of Antenna Carotenoid and Retinal in the Light-Driven Pumps of Salinibacter Ruber and Gloeobacter Violaceus

Xanthorhodopsin is a light-driven proton pump like bacteriorhodopsin, but contains a second chromophore, salinixanthin, a carotenoid. Action spectra for transport and fluorescence of the retinal upon excitation of salinixanthin, as well as femtosecond kinetics of carotenoid fluorescence, indicate that the carotenoid functions as an antenna to the retinal, with ca. 50% efficiency for excited-state energy transfer. The resulting charge redistribution in the retinal and the local electric field produced, in the excited state and in the primary stable photoproduct K, in turn, causes distinct electrochromic shifts in the carotenoid spectrum. The close interaction of carotenoid and retinal suggests that the two chromophores are in proximity. Tight binding of the carotenoid, as indicated by its sharpened vibration bands and intense induced circular dichroism in the visible, is removed in the absence of the retinal, and restored upon reconstitution with retinal or retinal analogues. The crystallographic structure of the xanthorhodopsin at 1.9 A resolution identifies the location of the chromophores. The structural model from x-ray diffraction reveals that the ring moieties of the retinal and the carotenoid are at nearly van der Waals distance from one another. Salinixanthin and one of the native carotenoids of Gloeobacter, echinenone, bind to gloeorhodopsin expressed in E. coli, and the complex shows excited-state energy exchange much like xanthorhodopsin. Protein sequences and mutational studies in Gloeobacter indicate that binding of the carotenoid depends on a conserved glycine at the keto-ring, which is a tryptophan in bacteriorhodopsin and other archaeal rhodopsins. On this basis, numerous otherwise unrelated eubacterial rhodopsins are predicted to contain light-harvesting carotenoids.

Photoaffinity Labeling the Propofol Binding Site in GLIC

Propofol, an intravenous general anesthetic, produces many of its anesthetic effects in vivo by potentiating the responses of GABA type A receptors (GABAAR), members of the superfamily of pentameric ligand-gated ion channels (pLGICs) that contain anion-selective channels. Propofol also inhibits pLGICs containing cation-selective channels, including nicotinic acetylcholine receptors and GLIC, a prokaryotic proton-gated homologue from Gloeobacter violaceus. In the structure of GLIC cocrystallized with propofol at pH 4 (presumed open/desensitized states), propofol was localized to an intrasubunit pocket at the extracellular end of the transmembrane domain within the bundle of transmembrane α-helices (Nury, H, et al. (2011) Nature 469, 428–431). To identify propofol binding sites in GLIC in solution, we used a recently developed photoreactive propofol analogue (2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol or AziPm) that acts as an anesthetic in vivo and potentiates GABAAR in vitro. For GLIC expr...

Establishment of the reporter system for a thylakoid-lacking cyanobacterium, Gloeobacter violaceus PCC 7421

Gloeobacter violaceus PCC 7421 is considered, by molecular phylogenetic analyses, to be an early-branching cyanobacterium within the cyanobacterial clade. G. violaceus is the only known oxygenic photosynthetic organism that lacks thylakoid membranes. There is only one report on the development of a transformation system for G. violaceus [H. Guo, X. Xu, Prog. Nat. Sci. 14 (2004) 31–35] and further studies using the system have not been reported. In the present study, we succeeded in introducing an expression vector (pKUT1121) derived from a broad-host-range plasmid, RSF1010, into G. violaceus by conjugation. The frequency of transformation of our system is significantly higher than that described in the previous report. In addition, luciferase heterologously expressed in G. violaceus functioned as a reporter. The established system will promote the molecular genetic studies on G. violaceus.

Modeling the Closed and Open State Conformations of the GABAA Ion Channel - Plausible Structural Insights for Channel Gating

Recent disclosure of high resolution crystal structures of Gloeobacter violaceus (GLIC) in open state and Erwinia chrysanthemii (ELIC) in closed state provides newer avenues to advance our knowledge and understanding of the physiologically and pharmacologically important ionotropic GABA(A) ion channel. The present modeling study envisions understanding the complex molecular transitions involved in ionic conductance, which were not evident in earlier disclosed homology models. In particular, emphasis was put on understanding the structural basis of gating, gating transition from the closed to the open state on an atomic scale. Homology modeling of two different physiological states of GABA(A) was carried out using their respective templates. The ability of induced fit docking in breaking the critical inter residue salt bridge (Glu155β(2) and Arg207β(2)) upon endogenous GABA docking reflects the perceived side chain rearrangements that occur at the orthosteric site and consolidate the quality of the model. Biophysical calculations like electrostatic mapping, pore radius calculation, ion solvation profile, and normal-mode analysis (NMA) were undertaken to address pertinent questions like the following: How the change in state of the ion channel alters the electrostatic environment across the lumen; How accessible is the Cl(-) ion in the open state and closed state; What structural changes regulate channel gating. A "Twist to Turn" global motion evinced at the quaternary level accompanied by tilting and rotation of the M2 helices along the membrane normal rationalizes the structural transition involved in gating. This perceived global motion hints toward a conserved gating mechanism among pLGIC. To paraphrase, this modeling study proves to be a reliable framework for understanding the structure function relationship of the hitherto unresolved GABA(A) ion channel. The modeled structures presented herein not only reveal the structurally distinct conformational states of the GABA(A) ion channel but also explain the biophysical difference between the respective states.

The Importance of TM3-4 Loop Subdomains for Functional Reconstitution of Glycine Receptors by Independent Domains

Truncated glycine receptors that have been found in human patients suffering from the neuromotor disorder hyperekplexia or in spontaneous mouse models resulted in non-functional ion channels. Rescue of function experiments with the lacking protein portion expressed as a separate independent domain demonstrated restoration of glycine receptor functionality in vitro. This construct harbored most of the TM3-4 loop, TM4, and the C terminus and was required for concomitant transport of the truncated α1 and the complementation domain from the endoplasmic reticulum toward the cell surface, thereby enabling complex formation of functional glycine receptors. Here, the complementation domain was stepwise truncated from its N terminus in the TM3-4 loop. Truncation of more than 49 amino acids led again to loss of functionality in the receptor complex expressed from two independent domain constructs. We identified residues 357-418 in the intracellular TM3-4 loop as being required for reconstitution of functional glycine-gated channels. All complementation constructs showed cell surface protein expression and correct orientation according to glycine receptor topology. Moreover, we demonstrated that the truncations did not result in a decreased protein-protein interaction between both glycine receptor domains. Rather, deletions of more than 49 amino acids abolished conformational changes necessary for ion channel opening. When the TM3-4 loop subdomain harboring residues 357-418 was expressed as a third independent construct together with the truncated N-terminal and C-terminal glycine receptor domains, functionality of the glycine receptor was again restored. Thus, residues 357-418 represent an important determinant in the process of conformational rearrangements following ligand binding resulting in channel opening.

Conformational Transitions Underlying Pore Opening and Desensitization in Membrane-embedded Gloeobacter violaceus Ligand-gated Ion Channel (GLIC)

Direct structural insight into the mechanisms underlying activation and desensitization remain unavailable for the pentameric ligand-gated channel family. Here, we report the structural rearrangements underlying gating transitions in membrane-embedded GLIC, a prokaryotic homologue, using site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. We particularly probed the conformation of pore-lining second transmembrane segment (M2) under conditions that favor the closed and the ligand-bound desensitized states. The spin label mobility, intersubunit spin-spin proximity, and the solvent-accessibility parameters in the two states clearly delineate the underlying protein motions within M2. Our results show that during activation the extracellular hydrophobic region undergoes major changes involving an outward translational movement, away from the pore axis, leading to an increase in the pore diameter, whereas the lower end of M2 remains relatively immobile. Most notably, during desensitization, the intervening polar residues in the middle of M2 move closer to form a solvent-occluded barrier and thereby reveal the location of a distinct desensitization gate. In comparison with the crystal structure of GLIC, the structural dynamics of the channel in a membrane environment suggest a more loosely packed conformation with water-accessible intrasubunit vestibules penetrating from the extracellular end all the way to the middle of M2 in the closed state. These regions have been implicated to play a major role in alcohol and drug modulation. Overall, these findings represent a key step toward understanding the fundamentals of gating mechanisms in this class of channels.

Microbial Proteomics: Archaea to Cyanobacteria

This Journal is Korean Society of Photoscience 2012 C 57 Thermococcus onnurineus NA1, sulfur reducing hyperthermophilic archaeon, is capable of H ‐ 2 producing growth and ‐ considered to be hydrogenogenic carboxydotrophy. Utilization of formate as a sole energy source has been well studied in T. onnurineus NA1. However, whether formate could be used as its carbon source remains unanswered question. To obtain a global view on the metabolic characteristics in H2 producing growth, the quantitative proteome analysis of ‐ T. onnurineus NA1 grown on formate, CO and starch was performed by combining 1DSDS PAGE with nano UPLCMS ‐ ‐ E method. Our data propose that CO could be utilized as its sole energy source via electron transport mechanism to produce H2. Additionally, our data propose that CO2 produced from catabolic pathway or CO oxidation by CODH and CO‐ induced hydrogenase activities could subsequently be assimilated into the organic carbon. Overall, proteomic comparison of formate and COgrown cells with starch grown cells revealed that one carbon compound such as formate and CO ‐ ‐ ‐ could be utilized as an efficient substrate to provide cellular carbon and/or energy in T. onnurineus NA1. Unicellular Gloeobacter violaceus PCC 7421 is a Gramnegative photoautotrophic cyanobacterium. ‐ Gloeobacter violaceus PCC 7421 was divided at very early point of the phylogenetic tree and contained very primitive and unusual features like lack of thylakoidal membrane and missing of some photosystem components. In this study we performed proteomic analysis of Gloeobacter violaceus PCC 7421 through the following two approaches. First, we separate the total, soluble and insoluble fractions of Gloeobacter violaceus PCC 7421 and performed the twodimensional ‐ electrophoresis (2DE). Second, insoluble fractions were analyzed by FTICR mass spectrometer. After 2DE separation ‐ ‐ ‐ in gel digested spots were identified by the MALDI/TOF/TOF analysis about 115, 73 and 21 proteins were identified ‐ from total, soluble and insoluble fractions, respectively. Also insoluble fraction was digested using both chemical and tryptic in solution digestion methods to obtain the more peptides with transmembrane regions. Following the FTICR ‐ ‐ analysis 785 proteins were identified and among them 139 proteins with 1 or more (~14) transmembrane domains were analyzed using TMHMM program. Considering the number of theoretical proteins (965) with 1~ TM about 14.4% of that proteins were found. Through these proteomic analyses we could determine the protein preparation methods and provide the 2DE map of ‐ Gloeobacter violaceus PCC 7421. Microbial Proteomics: Archaea to Cyanobacteria

A locally closed conformation of a bacterial pentameric proton-gated ion channel

Pentameric ligand-gated ion channels mediate signal transduction through conformational transitions between closed-pore and open-pore states. To stabilize a closed conformation of GLIC, a bacterial proton-gated homolog from Gloeobacter violaceus whose open structure is known, we separately generated either four cross-links or two single mutations. We found all six mutants to be in the same 'locally closed' conformation using X-ray crystallography, sharing most of the features of the open form but showing a locally closed pore as a result of a concerted bending of all of its M2 helices. The mutants adopt several variant conformations of the M2-M3 loop, and in all cases an interacting lipid that is observed in the open form disappears. A single cross-linked mutant is functional, according to electrophysiology, and the locally closed structure of this mutant indicates that it has an increased flexibility. Further cross-linking, accessibility and molecular dynamics data suggest that the locally closed form is a functionally relevant conformation that occurs during allosteric gating transitions.

Desensitization Mechanism in Prokaryotic Ligand-gated Ion Channel

Crystal structures of Gloeobacter violaceus ligand-gated ion channel (GLIC), a proton-gated prokaryotic homologue of pentameric ligand-gated ion channel (LGIC) from G. violaceus, have provided high-resolution models of the channel architecture and its role in selective ion conduction and drug binding. However, it is still unclear which functional states of the LGIC gating scheme these crystal structures represent. Much of this uncertainty arises from a lack of thorough understanding of the functional properties of these prokaryotic channels. To elucidate the molecular events that constitute gating, we have carried out an extensive characterization of GLIC function and dynamics in reconstituted proteoliposomes by patch clamp measurements and EPR spectroscopy. We find that GLIC channels show rapid activation upon jumps to acidic pH followed by a time-dependent loss of conductance because of desensitization. GLIC desensitization is strongly coupled to activation and is modulated by voltage, permeant ions, pore-blocking drugs, and membrane cholesterol. Many of these properties are parallel to functions observed in members of eukaryotic LGIC. Conformational changes in loop C, measured by site-directed spin labeling and EPR spectroscopy, reveal immobilization during desensitization analogous to changes in LGIC and acetylcholine binding protein. Together, our studies suggest conservation of mechanistic aspects of desensitization among LGICs of prokaryotic and eukaryotic origin.

Binding Site and Affinity Prediction of General Anesthetics to Protein Targets Using Docking

The protein targets for general anesthetics remain unclear. A tool to predict anesthetic binding for potential binding targets is needed. In this study, we explored whether a computational method, AutoDock, could serve as such a tool. High-resolution crystal data of water-soluble proteins (cytochrome C, apoferritin, and human serum albumin), and a membrane protein (a pentameric ligand-gated ion channel from Gloeobacter violaceus [GLIC]) were used. Isothermal titration calorimetry (ITC) experiments were performed to determine anesthetic affinity in solution conditions for apoferritin. Docking calculations were performed using DockingServer with the Lamarckian genetic algorithm and the Solis and Wets local search method (http://www.dockingserver.com/web). Twenty general anesthetics were docked into apoferritin. The predicted binding constants were compared with those obtained from ITC experiments for potential correlations. In the case of apoferritin, details of the binding site and their interactions were compared with recent cocrystallization data. Docking calculations for 6 general anesthetics currently used in clinical settings (isoflurane, sevoflurane, desflurane, halothane, propofol, and etomidate) with known 50% effective concentration (EC(50)) values were also performed in all tested proteins. The binding constants derived from docking experiments were compared with known EC(50) values and octanol/water partition coefficients for the 6 general anesthetics. All 20 general anesthetics docked unambiguously into the anesthetic binding site identified in the crystal structure of apoferritin. The binding constants for 20 anesthetics obtained from the docking calculations correlate significantly with those obtained from ITC experiments (P = 0.04). In the case of GLIC, the identified anesthetic binding sites in the crystal structure are among the docking predicted binding sites, but not the top ranked site. Docking calculations suggest a most probable binding site located in the extracellular domain of GLIC. The predicted affinities correlated significantly with the known EC(50) values for the 6 frequently used anesthetics in GLIC for the site identified in the experimental crystal data (P = 0.006). However, predicted affinities in apoferritin, human serum albumin, and cytochrome C did not correlate with these 6 anesthetics' known experimental EC(50) values. A weak correlation between the predicted affinities and the octanol/water partition coefficients was observed for the sites in GLIC. We demonstrated that anesthetic binding sites and relative affinities can be predicted using docking calculations in an automatic docking server (AutoDock) for both water-soluble and membrane proteins. Correlation of predicted affinity and EC(50) for 6 frequently used general anesthetics was only observed in GLIC, a member of a protein family relevant to anesthetic mechanism.

Unique Properties vs. Common Themes: The Atypical Cyanobacterium Gloeobacter violaceus PCC 7421 is Capable of State Transitions and Blue-Light-Induced Fluorescence Quenching

The atypical unicellular cyanobacterium Gloeobacter violaceus PCC 7421, which diverged very early during the evolution of cyanobacteria, can be regarded as a key organism for understanding many structural, functional, regulatory and evolutionary aspects of oxygenic photosynthesis. In the present work, the performance of two basic photosynthetic adaptation/protection mechanisms, common to all other oxygenic photoautrophs, had been challenged in this ancient cyanobacterium which lacks thylakoid membranes: state transitions and non-photochemical fluorescence quenching. Both low temperature fluorescence spectra and room temperature fluorescence transients show that G. violaceus is capable of performing state transitions similar to evolutionarily more recent cyanobacteria, being in state 2 in darkness and in state 1 upon illumination by weak blue or far-red light. Compared with state 2, variable fluorescence yield in state 1 is strongly enhanced (almost 80%), while the functional absorption cross-section of PSII is only increased by 8%. In contrast to weak blue light, which enhances fluorescence yield via state 1 formation, strong blue light reversibly quenches Chl fluorescence in G. violaceus. This strongly suggests regulated heat dissipation which is triggered by the orange carotenoid protein whose presence was directly proven by immunoblotting and mass spectrometry in this primordial cyanobacterium. The results are discussed in the framework of cyanobacterial evolution.

The Molecular Mechanism for the Dual Alcohol Modulation of Cys-Loop Receptors

Despite high sequence identity, pentameric ligand-gated ion channels exhibit remarkable diversity in function with anionic/cationic channels that are either potentiated or inhibited by allosteric ligands. The recently available structures of bacterial homologs of Cys-loop receptors provide an excellent framework for understanding this allosteric modulation and function, but the modeling can be complex; our earlier simulations of the prokaryotic anionic glycine receptor (GlyR) suggest inter-subunit binding for ethanol (Murail, Biophys J 100, 1642, 2011), which at first sight appears to be incompatible with the experimental Gloeobacter violaceus (GLIC) ligand-gated ion channel structure showing binding intra-subunit. Here, we present new simulations of GLIC that confirm the occurrence of multiple binding sites by showing intra-subunit binding for ethanol. By experimentally introducing the single-site F238A mutation in GLIC we can turn it into a highly ethanol-sensitive channel (Howard, PNAS 108, 12149, 2011), similar to GlyR, and simulations of the mutated species confirm the occurrence of multiple binding sites. To critically test the results, we performed extensive docking and free energy calculations to identify alcohol-binding sites and determine their affinity. In the wild-type GLIC, short alcohols preferentially bind intra-subunit, with a very weak binding site inter-subunit. However, with the F238A mutation the inter-subunit site achieves a significantly lower free energy, and even becomes the highest-affinity site in the channel for some alcohols. These results suggest a new model for pentameric ligand-gated channel potentiation and inhibition, where the intra-subunit cavity would control inhibition, and the inter-subunit cavity potentiation. The possibility of allosteric ligands interacting with both cavities - or even a single molecule stretching from one site to the other - offers an attractive explanation for the complex functional dynamics of ligand-gated ion channels as well as an potential explain for the alcohol cutoff effect.

Allosteric Transition and Pharmacological Modulation of Pentameric Channel-Receptors: The Case of General Anesthetics

General anaesthetics have enjoyed long and widespread use but their molecular mechanism of action remains poorly understood. There is good evidence that their principal targets are pentameric ligand-gated ion channels (pLGICs) such as inhibitory GABAA (γ-aminobutyric acid receptor A) and excitatory nicotinic acetylcholine receptors (nAChRs), which are respectively potentiated and inhibited by these allosteric effectors. The bacterial homologue from Gloeobacter violaceus (GLIC), whose X-ray structure was recently solved, is also sensitive to clinical concentrations of general anaesthetics. We recently solved the crystal structures of the complexes propofol/GLIC and desflurane/GLIC. These reveal a common general-anaesthetic binding site which pre-exists in the apo-structure in the upper part of the transmembrane domain of each protomer. Both molecules establish van der Waals interactions with the protein; propofol binds at the entrance of the cavity whereas the smaller, more flexible, desflurane binds deeper inside. Mutations of some amino acids lining the binding site profoundly alter the ionic response of GLIC to protons, and affect general-anaesthetic pharmacology. Simulations of molecular dynamics, performed on the wild type and two GLIC mutants, highlight differences in mobility of propofol in its binding site and help to explain these effects. These data provide a novel structural framework for the design of general anaesthetics and of allosteric modulators of brain pLGICs, including nAChRs. In addition, they give insights into the gating mechanism occurring in this family of channel, that involve transmission of conformational reorganization from the extracellular domain to the transmembrane domain

X-Ray Structures of General Anaesthetics Bound to a Pentameric Ligand-Gated Ion Channel

General anaesthetics have enjoyed long and widespread use but their molecular mechanism of action remains poorly understood. There is good evidence that their principal targets are pentameric ligand-gated ion channels (pLGICs) such as inhibitory GABAA (γ-aminobutyric acid) receptors and excitatory nicotinic acetylcholine receptors, which are respectively potentiated and inhibited by general anaesthetics. The bacterial homologue from Gloeobacter violaceus (GLIC), whose X-ray structure was recently solved, is also sensitive to clinical concentrations of general anaesthetics. Here we describe the crystal structures of the complexes propofol/GLIC and desflurane/GLIC. These reveal a common general-anaesthetic binding site, which pre-exists in the apo-structure in the upper part of the transmembrane domain of each protomer. Both molecules establish van der Waals interactions with the protein; propofol binds at the entrance of the cavity whereas the smaller, more flexible, desflurane binds deeper inside. Mutations of some amino acids lining the binding site profoundly alter the ionic response of GLIC to protons, and affect its general-anaesthetic pharmacology. Molecular dynamics simulations, performed on the wild type and two GLIC mutants, highlight differences in mobility of propofol in its binding site and help to explain these effects. These data provide a novel structural framework for the design of general anaesthetics and of allosteric modulators of brain pLGICs. Nury H, Van Renterghem C, Weng Y, Tran A, Dufresne V, Baaden M, Changeux JP, Sonner JM, Delarue M, Corringer PJ. Nature 469, 428-431 (2011).