The Location and Nature of General Anesthetic Binding Sites on the Active Conformation of Firefly Luciferase; A Time Resolved Photolabeling Study

Firefly luciferase is one of the few soluble proteins that is acted upon by a wide variety of general anesthetics and alcohols; they inhibit the ATP–driven production of light. We have used time–resolved photolabeling to locate the binding sites of alcohols during the initial light output, some 200 ms after adding ATP. The photolabel 3-azioctanol inhibited the initial light output with an IC50 of 200 µM, close to its general anesthetic potency. Photoincorporation of [3H]3-azioctanol into luciferase was saturable but weak. It was enhanced 200 ms after adding ATP but was negligible minutes later. Sequencing of tryptic digests by HPLC–MSMS revealed a similar conformation–dependence for photoincorporation of 3-azioctanol into Glu-313, a residue that lines the bottom of a deep cleft (vestibule) whose outer end binds luciferin. An aromatic diazirine analog of benzyl alcohol with broader side chain reactivity reported two sites. First, it photolabeled two residues in the vestibule, Ser-286 and Ile-288, both of which are implicated with Glu-313 in the conformation change accompanying activation. Second, it photolabeled two residues that contact luciferin, Ser-316 and Ser-349. Thus, time resolved photolabeling supports two mechanisms of action. First, an allosteric one, in which anesthetics bind in the vestibule displacing water molecules that are thought to be involved in light output. Second, a competitive one, in which anesthetics bind isosterically with luciferin. This work provides structural evidence that supports the competitive and allosteric actions previously characterized by kinetic studies

Recognition of Anesthetic Barbiturates by a Protein Binding Site: A High Resolution Structural Analysis

Barbiturates potentiate GABA actions at the GABAA receptor and act as central nervous system depressants that can induce effects ranging from sedation to general anesthesia. No structural information has been available about how barbiturates are recognized by their protein targets. For this reason, we tested whether these drugs were able to bind specifically to horse spleen apoferritin, a model protein that has previously been shown to bind many anesthetic agents with affinities that are closely correlated with anesthetic potency. Thiopental, pentobarbital, and phenobarbital were all found to bind to apoferritin with affinities ranging from 10–500 µM, approximately matching the concentrations required to produce anesthetic and GABAergic responses. X-ray crystal structures were determined for the complexes of apoferritin with thiopental and pentobarbital at resolutions of 1.9 and 2.0 Å, respectively. These structures reveal that the barbiturates bind to a cavity in the apoferritin shell that also binds haloalkanes, halogenated ethers, and propofol. Unlike these other general anesthetics, however, which rely entirely upon van der Waals interactions and the hydrophobic effect for recognition, the barbiturates are recognized in the apoferritin site using a mixture of both polar and nonpolar interactions. These results suggest that any protein binding site that is able to recognize and respond to the chemically and structurally diverse set of compounds used as general anesthetics is likely to include a versatile mixture of both polar and hydrophobic elements

Intravenous Hypnotic Agents: From Binding Sites to Loss of Consciousness

All the intravenous hypnotic drugs important for clinical anesthesiology reversibly unsettle functional brain networks, in order to undermine the information transfer on which consciousness depends. Three classes of intravenous hypnotic drugs are the most used nowadays: the carboxylated imidazole derivate propofol, the short-acting benzodiazepine midazolam, and the barbiturates, which show action on GABAA Receptors, potentiating gamma-aminobutyric acid (GABA) action. The dissociative agent ketamine, instead, mainly exerts its effects by reversibly blocking the activity of N-methyl-D-aspartate receptors while the most recent dexmedetomidine is an alpha-2 adrenergic receptor agonist. Nevertheless, other receptors are also involved in anesthesia determining, that is voltage-gated and ligand-gated ion channels and it is probable that each intravenous hypnotic agent alters neuronal activity acting at different levels and at multiple sites in a way not yet entirely clear

Major phytoplasma diseases of forest and urban trees

In the northern hemisphere, yellows, witches’ broom, and decline diseases of several forest and urban tree species are widespread and of considerable economic and ecological significance. Elm (Ulmus spp.) and alder (Alnus spp.) are affected by elm yellows (EY) and alder yellows (ALY), respectively. These diseases are mainly associated with the presence of closely related phytoplasmas, the EY agent ‘Candidatus Phytoplasma ulmi’ and the ALY agent, which are members of the EY or 16SrV group, subgroups 16SrV-A and 16SrV-C, respectively. Ash (Fraxinus spp.) is affected by ash yellows, a disease which occurs mainly in North America and is associated with the presence of ‘Candidatus Phytoplasma fraxini’, a member of subgroup 16SrVII-A. Poplar (Populus spp.), sandal (Santalum album), paulownia (Paulownia spp.), and mulberry (Morus spp.) are affected by yellows diseases associated with phytoplasmas of different 16SrI subgroups. Several species of conifers are affected by yellows and witches’ broom diseases associated with phytoplasmas belonging to at least five taxonomic groups (16SrI, 16SrIII, 16SrVI, 16SrIX, and 16SrXXI) and several different subgroups. A number of urban tree species grown in the Sabana de Bogotà (Colombia) are affected by decline diseases which are primarily associated with 16SrI and 16SrVII phytoplasmas. This chapter summarizes the current knowledge of major phytoplasma diseases of forest and urban trees grown in the northern hemisphere

Hydrocarbon molar water solubility predicts NMDA vs. GABAA receptor modulation

BACKGROUND: Many anesthetics modulate 3-transmembrane (such as NMDA) and 4-transmembrane (such as GABA(A)) receptors. Clinical and experimental anesthetics exhibiting receptor family specificity often have low water solubility. We hypothesized that the molar water solubility of a hydrocarbon could be used to predict receptor modulation in vitro. METHODS: GABA(A) (α(1)β(2)γ(2s)) or NMDA (NR1/NR2A) receptors were expressed in oocytes and studied using standard two-electrode voltage clamp techniques. Hydrocarbons from 14 different organic functional groups were studied at saturated concentrations, and compounds within each group differed only by the carbon number at the ω-position or within a saturated ring. An effect on GABA(A) or NMDA receptors was defined as a 10% or greater reversible current change from baseline that was statistically different from zero. RESULTS: Hydrocarbon moieties potentiated GABA(A) and inhibited NMDA receptor currents with at least some members from each functional group modulating both receptor types. A water solubility cut-off for NMDA receptors occurred at 1.1 mM with a 95% CI = 0.45 to 2.8 mM. NMDA receptor cut-off effects were not well correlated with hydrocarbon chain length or molecular volume. No cut-off was observed for GABA(A) receptors within the solubility range of hydrocarbons studied. CONCLUSIONS: Hydrocarbon modulation of NMDA receptor function exhibits a molar water solubility cut-off. Differences between unrelated receptor cut-off values suggest that the number, affinity, or efficacy of protein-hydrocarbon interactions at these sites likely differ