Roles of transmembrane domain 2 and the first intracellular loop in human noradrenaline transporter function: pharmacological and SCAM analysis

The aim was to investigate the roles of transmembrane domain 2 and the adjacent region of the first intracellular loop in determining human noradrenaline transporter (hNET) function by pharmacological and substituted-cysteine accessibility method (SCAM) analyses. It was first necessary to establish a suitable background NET for SCAM. Alanine mutants of endogenous hNET cysteines, hC86A, hC131A and hC339A, were examined and showed no marked effects on expression or function. hNET and the mutants were also resistant to methanethiosulfonate (MTS), ethylammonium (MTSEA) and MTStrimethylammonium (MTSET). Hence, wild-type hNET is an appropriate background for production of cysteine mutants for SCAM. Pharmacological investigation showed that all mutants except hT99C and hL109C showed reduced cell-surface expression, while all except hM107C showed a reduction in functional activity. The mutations did not markedly affect the apparent affinities of substrates, but apparent affinities of cocaine were decreased 7-fold for hP97C and 10-fold for hF101C and increased 12-fold for hY98C. [H-3]Nisoxetine binding affinities were decreased 13-fold for hP97C and 5-fold for hF101C. SCAM analysis revealed that only hL102C was sensitive to 1.25 mM MTSEA, and this sensitivity was protected by noradrenaline, nisoxetine and cocaine. The results suggest that this region of hNET is important for interactions with antidepressants and cocaine, but it is probably not involved in substrate translocation mechanisms.

A picrotoxin-specific conformational change in the glycine receptor M2-M3 loop.

The external loop linking the M2 and M3 transmembrane domains is crucial for coupling agonist binding to channel gating in the glycine receptor chloride channel (GlyR). A substituted cysteine accessibility scan previously showed that glycine activation increased the surface accessibility of 6 contiguous residues (Arg(271) Lys(276)) toward the N-terminal end of the homomeric alpha 1 GlyR M2 - M3 loop. In the present study we used a similar approach to determine whether the allosteric antagonist, picrotoxin, could impose conformational changes to this domain that cannot be induced by varying agonist concentrations alone. Picrotoxin slowed the reaction rate of a sulfhydryl-containing compound ( MTSET) with A272C, S273C, and L274C. Before interpreting this as a picrotoxin-specific conformational change, it was necessary to eliminate the possibility of steric competition between picrotoxin and MTSET. Accordingly, we showed that picrotoxin and the structurally unrelated blocker, bilobalide, were both trapped in the R271C GlyR in the closed state and that a point mutation to the pore-lining Thr(6') residue abolished inhibition by both compounds. We also demonstrated that the picrotoxin dissociation rate was linearly related to the channel open probability. These observations constitute a strong case for picrotoxin binding in the pore. We thus conclude that the picrotoxin-specific effects on the M2 - M3 loop are mediated allosterically. This suggests that the M2 - M3 loop responds differently to the occupation of different binding sites.

The adsorption of water in finite carbon pores

Grand canonical Monte Carlo simulations were applied to the adsorption of SPCE model water in finite graphitic pores with different configurations of carbonyl functional groups on only one surface and several pore sizes. It was found that almost all finite pores studied exhibit capillary condensation behaviour preceded by adsorption around the functional groups. Desorption showed the reverse transitions from a filled to a near empty pore resulting in a clear hysteresis loop in all pores except for some of the configurations of the 1.0nm pore. Carbonyl configurations had a strong effect on the filling pressure of all pores except, in some cases, in 1.0nm pores. A decrease in carbonyl neighbour density would result in a higher filling pressure. The emptying pressure was negligibly affected by the configuration of functional groups. Both the filling and emptying pressures increased with increasing pore size but the effect on the emptying pressure was much less. At pressures lower than the pore filling pressure, the adsorption of water was shown to have an extremely strong dependence on the neighbour density with adsorption changing from Type IV to Type III to linear as the neighbour density decreased. The isosteric heat was also calculated for these configurations to reveal its strong dependence on the neighbour density. These results were compared with literature experimental results for water and carbon black and found to qualitatively agree.

First-principles quantum simulations of dissociation of molecular condensates: Atom correlations in momentum space

We investigate the quantum many-body dynamics of dissociation of a Bose-Einstein condensate of molecular dimers into pairs of constituent bosonic atoms and analyze the resulting atom-atom correlations. The quantum fields of both the molecules and atoms are simulated from first principles in three dimensions using the positive-P representation method. This allows us to provide an exact treatment of the molecular field depletion and s-wave scattering interactions between the particles, as well as to extend the analysis to nonuniform systems. In the simplest uniform case, we find that the major source of atom-atom decorrelation is atom-atom recombination which produces molecules outside the initially occupied condensate mode. The unwanted molecules are formed from dissociated atom pairs with nonopposite momenta. The net effect of this process-which becomes increasingly significant for dissociation durations corresponding to more than about 40% conversion-is to reduce the atom-atom correlations. In addition, for nonuniform systems we find that mode mixing due to inhomogeneity can result in further degradation of the correlation signal. We characterize the correlation strength via the degree of squeezing of particle number-difference fluctuations in a certain momentum-space volume and show that the correlation strength can be increased if the signals are binned into larger counting volumes.

Mimicking the action of GroEL in molecular dynamics simulations: Application to the refinement of protein structures

Bacterial chaperonin, GroEL, together with its co-chaperonin, GroES, facilitates the folding of a variety of polypeptides. Experiments suggest that GroEL stimulates protein folding by multiple cycles of binding and release. Misfolded proteins first bind to an exposed hydrophobic surface on GroEL. GroES then encapsulates the substrate and triggers its release into the central cavity of the GroEL/ES complex for folding. In this work, we investigate the possibility to facilitate protein folding in molecular dynamics simulations by mimicking the effects of GroEL/ES namely, repeated binding and release, together with spatial confinement. During the binding stage, the (metastable) partially folded proteins are allowed to attach spontaneously to a hydrophobic surface within the simulation box. This destabilizes the structures, which are then transferred into a spatially confined cavity for folding. The approach has been tested by attempting to refine protein structural models generated using the ROSETTA procedure for ab initio structure prediction. Dramatic improvements in regard to the deviation of protein models from the corresponding experimental structures were observed. The results suggest that the primary effects of the GroEL/ES system can be mimicked in a simple coarse-grained manner and be used to facilitate protein folding in molecular dynamics simulations. Furthermore, the results Sur port the assumption that the spatial confinement in GroEL/ES assists the folding of encapsulated proteins.

Molecular dynamics simulations of the hydrophobin SC3 at a hydrophobic/hydrophilic interface

Hydrophobins are small (similar to 100 aa) proteins that have an important role in the growth and development of mycelial fungi. They are surface active and, after secretion by the fungi, self-assemble into amphipathic membranes at hydrophobic/hydrophilic interfaces, reversing the hydrophobicity of the surface. In this study, molecular dynamics simulation techniques have been used to model the process by which a specific class I hydrophobin, SC3, binds to a range of hydrophobic/ hydrophilic interfaces. The structure of SC3 used in this investigation was modeled based on the crystal structure of the class II hydrophobin HFBII using the assumption that the disulfide pairings of the eight conserved cysteine residues are maintained. The proposed model for SC3 in aqueous solution is compact and globular containing primarily P-strand and coil structures. The behavior of this model of SC3 was investigated at an air/water, an oil/water, and a hydrophobic solid/water interface. It was found that SC3 preferentially binds to the interfaces via the loop region between the third and fourth cysteine residues and that binding is associated with an increase in a-helix formation in qualitative agreement with experiment. Based on a combination of the available experiment data and the current simulation studies, we propose a possible model for SC3 self-assembly on a hydrophobic solid/water interface.

The molecular basis of temperature compensation in the Arabidopsis circadian clock

Circadian clocks maintain robust and accurate timing over a broad range of physiological temperatures, a characteristic termed temperature compensation. In Arabidopsis thaliana, ambient temperature affects the rhythmic accumulation of transcripts encoding the clock components TIMING OF CAB EXPRESSION1 (TOC1), GIGANTEA (GI), and the partially redundant genes CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). The amplitude and peak levels increase for TOC1 and GI RNA rhythms as the temperature increases (from 17 to 27 degrees C), whereas they decrease for LHY. However, as temperatures decrease ( from 17 to 12 degrees C), CCA1 and LHY RNA rhythms increase in amplitude and peak expression level. At 27 degrees C, a dynamic balance between GI and LHY allows temperature compensation in wild-type plants, but circadian function is impaired in Ihy and gi mutant plants. However, at 12 degrees C, CCA1 has more effect on the buffering mechanism than LHY, as the cca1 and gi mutations impair circadian rhythms more than Ihy at the lower temperature. At 17 degrees C, GI is apparently dispensable for free-running circadian rhythms, although partial GI function can affect circadian period. Numerical simulations using the interlocking-loop model show that balancing LHY/CCA1 function against GI and other evening-expressed genes can largely account for temperature compensation in wild-type plants and the temperature-specific phenotypes of gi mutants.

Stochastic modelling and simulations for solute transport in porous media

A stochastic model for solute transport in aquifers is studied based on the concepts of stochastic velocity and stochastic diffusivity. By applying finite difference techniques to the spatial variables of the stochastic governing equation, a system of stiff stochastic ordinary differential equations is obtained. Both the semi-implicit Euler method and the balanced implicit method are used for solving this stochastic system. Based on the Karhunen-Loeve expansion, stochastic processes in time and space are calculated by means of a spatial correlation matrix. Four types of spatial correlation matrices are presented based on the hydraulic properties of physical parameters. Simulations with two types of correlation matrices are presented.

The programming language python in earth system simulations

-scale vary from a planetary scale and million years for convection problems to 100km and 10 years for fault systems simulations. Various techniques are in use to deal with the time dependency (e.g. Crank-Nicholson), with the non-linearity (e.g. Newton-Raphson) and weakly coupled equations (e.g. non-linear Gauss-Seidel). Besides these high-level solution algorithms discretization methods (e.g. finite element method (FEM), boundary element method (BEM)) are used to deal with spatial derivatives. Typically, large-scale, three dimensional meshes are required to resolve geometrical complexity (e.g. in the case of fault systems) or features in the solution (e.g. in mantel convection simulations). The modelling environment escript allows the rapid implementation of new physics as required for the development of simulation codes in earth sciences. Its main object is to provide a programming language, where the user can define new models and rapidly develop high-level solution algorithms. The current implementation is linked with the finite element package finley as a PDE solver. However, the design is open and other discretization technologies such as finite differences and boundary element methods could be included. escript is implemented as an extension of the interactive programming environment python (see www.python.org). Key concepts introduced are Data objects, which are holding values on nodes or elements of the finite element mesh, and linearPDE objects, which are defining linear partial differential equations to be solved by the underlying discretization technology. In this paper we will show the basic concepts of escript and will show how escript is used to implement a simulation code for interacting fault systems. We will show some results of large-scale, parallel simulations on an SGI Altix system. Acknowledgements: Project work is supported by Australian Commonwealth Government through the Australian Computational Earth Systems Simulator Major National Research Facility, Queensland State Government Smart State Research Facility Fund, The University of Queensland and SGI.

IT support for managing aircraft spares in a closed-loop supply chain

This paper reports on the development of elements of an e-supply chain management system for managing maintenance, repair and overhaul (MRO) relationships in the aerospace industry. A standard systems development methodology has been followed to produce a process model (i.e. the AMSCR model); an information model (i.e. business rules) and a computerised information management capability (i.e. automated optimisation). The proof of concept for this web-based MRO supply chain system has been established through the collaboration with a sample of the different types of supply chain members. The proven benefit is a reduction in the stock-holding costs for the whole supply chain whilst also minimising non-flying time of the aircraft that the supply chain supports. This type of system is now vital in an industry that has continuously decreasing profit margins, which in turn means pressure to reduce servicing times and increase the interval between maintenance actions.