Signal Transduction Pathways in the Pentameric Ligand-Gated Ion Channels

The mechanisms of allosteric action within pentameric ligand-gated ion channels (pLGICs) remain to be determined. Using crystallography, site-directed mutagenesis, and two-electrode voltage clamp measurements, we identified two functionally relevant sites in the extracellular (EC) domain of the bacterial pLGIC from Gloeobacter violaceus (GLIC). One site is at the C-loop region, where the NQN mutation (D91N, E177Q, and D178N) eliminated inter-subunit salt bridges in the open-channel GLIC structure and thereby shifted the channel activation to a higher agonist concentration. The other site is below the C-loop, where binding of the anesthetic ketamine inhibited GLIC currents in a concentration dependent manner. To understand how a perturbation signal in the EC domain, either resulting from the NQN mutation or ketamine binding, is transduced to the channel gate, we have used the Perturbation-based Markovian Transmission (PMT) model to determine dynamic responses of the GLIC channel and signaling pathways upon initial perturbations in the EC domain of GLIC. Despite the existence of many possible routes for the initial perturbation signal to reach the channel gate, the PMT model in combination with Yen's algorithm revealed that perturbation signals with the highest probability flow travel either via the β1-β2 loop or through pre-TM1. The β1-β2 loop occurs in either intra- or inter-subunit pathways, while pre-TM1 occurs exclusively in inter-subunit pathways. Residues involved in both types of pathways are well supported by previous experimental data on nAChR. The direct coupling between pre-TM1 and TM2 of the adjacent subunit adds new insight into the allosteric signaling mechanism in pLGICs. © 2013 Mowrey et al

The benzene polycarboxylic acid (BPCA) pattern of wood pyrolyzed between 200°C and 1000°C

Environmental charcoals represent a poorly defined part of the black carbon (BC) combustion continuum and may differ widely in their chemical and physical properties, depending on combustion conditions and source material. The benzene polycarboxylic acid (BPCA) molecular marker method is well established to quantify the BC component in charcoal, soil and sediment, although observed variations between labs could stem from subtle differences in methods. The objectives of this study were to identify and improve potential sources of analytical uncertainty. The improved method was then used to qualitatively characterize wood charred at 200–1000 °C. One significant improvement of the BPCA method was to replace citric acid with phthalic acid as an internal standard, which is more stable in acidic solution and more similar to the target compounds. Also, including a soil reference material as a quality control in each analysis proved to be a robust tool to detect for variations in reproducibility. For the thermosequence, elemental O/C and H/C ratios typically decreased with temperature to 60.03 at 1000 °C, whereas BPCA concentrations peaked at 700 °C. With temperature B6CA proportions increased consistently (6–98%), except for a plateau at 250–500 °C. Thus, relative contributions of B6CA reflected the pyrolysis temperature and probably also the degree of condensation of the charcoals we investigated. Future work will show if our results can be directly related to charcoal produced under oxygen limited conditions, including charcoal formed at wildfires or so called biochar for agricultural use

General Anesthetics Predicted to Block the GLIC Pore with Micromolar Affinity

Although general anesthetics are known to modulate the activity of ligand-gated ion channels in the Cys-loop superfamily, there is at present neither consensus on the underlying mechanisms, nor predictive models of this modulation. Viable models need to offer quantitative assessment of the relative importance of several identified anesthetic binding sites. However, to date, precise affinity data for individual sites has been challenging to obtain by biophysical means. Here, the likely role of pore block inhibition by the general anesthetics isoflurane and propofol of the prokaryotic pentameric channel GLIC is investigated by molecular simulations. Microscopic affinities are calculated for both single and double occupancy binding of isoflurane and propofol to the GLIC pore. Computations are carried out for an open-pore conformation in which the pore is restrained to crystallographic radius, and a closed-pore conformation that results from unrestrained molecular dynamics equilibration of the structure. The GLIC pore is predicted to be blocked at the micromolar concentrations for which inhibition by isofluorane and propofol is observed experimentally. Calculated affinities suggest that pore block by propofol occurs at signifcantly lower concentrations than those for which inhibition is observed: we argue that this discrepancy may result from binding of propofol to an allosteric site recently identified by X-ray crystallography, which may cause a competing gain-of-function effect. Affinities of isoflurane and propofol to the allosteric site are also calculated, and shown to be 3 mM for isoflurane and for propofol; both anesthetics have a lower affinity for the allosteric site than for the unoccupied pore

Olefin cross metathesis and ring-closing metathesis in polymer chemistry

The use of olefin cross metathesis in preparing functional polymers, through either pre-functionalisation of monomers or post-polymerisation functionalisation is growing in both scope and breadth. The broad functional group tolerance of olefin metathesis offers a wealth of opportunities for introducing a broad range of functional groups into the polymer backbone, tuning polymer properties and expanding potential applications. Similarly, ring-closing metathesis offers the ability to tune the polymer macrostructure and microstructure to similar effect. In this review, we explore the importance of understanding selectivity in olefin cross metathesis in designing functional polymers, the manipulation of this reactivity to prepare (multi)functional polymers, and show how polymer systems can be constructed to favour ring closing and change backbone structure and properties

APBSmem: A Graphical Interface for Electrostatic Calculations at the Membrane

Electrostatic forces are one of the primary determinants of molecular interactions. They help guide the folding of proteins, increase the binding of one protein to another and facilitate protein-DNA and protein-ligand binding. A popular method for computing the electrostatic properties of biological systems is to numerically solve the Poisson-Boltzmann (PB) equation, and there are several easy-to-use software packages available that solve the PB equation for soluble proteins. Here we present a freely available program, called APBSmem, for carrying out these calculations in the presence of a membrane. The Adaptive Poisson-Boltzmann Solver (APBS) is used as a back-end for solving the PB equation, and a Java-based graphical user interface (GUI) coordinates a set of routines that introduce the influence of the membrane, determine its placement relative to the protein, and set the membrane potential. The software Jmol is embedded in the GUI to visualize the protein inserted in the membrane before the calculation and the electrostatic potential after completing the computation. We expect that the ease with which the GUI allows one to carry out these calculations will make this software a useful resource for experimenters and computational researchers alike. Three examples of membrane protein electrostatic calculations are carried out to illustrate how to use APBSmem and to highlight the different quantities of interest that can be calculated

DOARC - Distributed Open Access Reference Citations Service

DOARC (www.isn-oldenburg.de/projects/doarc2/ with its demonstator doarc.projects.isn-oldenburg.de) Distributed Open Access Reference Citations, is a new service under development to be served by DINI as part of its emerging OA-Network System (www.dini.de and www.dini.de/projekte/oa-netzwerk) and funded by the DFG (German Science Foundation DFG, www.dfg.de) which aims at creating an interactive reference index for scientific documents. Special emphasis is given to the Open Access (OA) documents posted by the present German OAI-PMH-Institutional Repositories at Universities and large Research Institutions. One part of it will be a citation-based user interface with tools for authors and readers. The general motivation behind DOARC is to serve add-on services with regard to citations and specically exploit and make use of the opportunities that the OA document world offers by its access to the full text documents. This will provide an extra benefit for both, authors and readers and thus boost the way to spread OA and thus in the end add to increase the rate of citations in an OA world. Specically, by DOARC authors will be given a tool, to ensure that they cite correctly, and that their document's references list will be extracted and added to the pool of DOARC citations. Readers will get a tool by which they can find a document relevant for them by browsing through citations and by a graphical tool which shows the 'content affinity' to other documents in the widely distributed pool of scientific OA-papers. We will exchange our checked metadata with other citation services and further the know-how for non-commercial citation services. In the interface the user will be able to see references with additional information of high value (enriched metadata). We are integrating the services into a wider European context by joining a new initiative organized by Alma Swan of Key Perspectives

DOARC - Distributed Open Access Reference Citations Service

DOARC (www.isn-oldenburg.de/projects/doarc2/ with its demonstator doarc.projects.isn-oldenburg.de) Distributed Open Access Reference Citations, is a new service under development to be served by DINI as part of its emerging OA-Network System (www.dini.de and www.dini.de/projekte/oa-netzwerk) and funded by the DFG (German Science Foundation DFG, www.dfg.de) which aims at creating an interactive reference index for scientific documents. Special emphasis is given to the Open Access (OA) documents posted by the present German OAI-PMH-Institutional Repositories at Universities and large Research Institutions. One part of it will be a citation-based user interface with tools for authors and readers. The general motivation behind DOARC is to serve add-on services with regard to citations and specically exploit and make use of the opportunities that the OA document world offers by its access to the full text documents. This will provide an extra benefit for both, authors and readers and thus boost the way to spread OA and thus in the end add to increase the rate of citations in an OA world. Specically, by DOARC authors will be given a tool, to ensure that they cite correctly, and that their document's references list will be extracted and added to the pool of DOARC citations. Readers will get a tool by which they can find a document relevant for them by browsing through citations and by a graphical tool which shows the 'content affinity' to other documents in the widely distributed pool of scientific OA-papers. We will exchange our checked metadata with other citation services and further the know-how for non-commercial citation services. In the interface the user will be able to see references with additional information of high value (enriched metadata). We are integrating the services into a wider European context by joining a new initiative organized by Alma Swan of Key Perspectives