Two Distinct Structures of Membrane-Associated Homodimers of GTP- and GDP-Bound KRAS4B Revealed by Paramagnetic Relaxation Enhancement

KRAS homo-dimerization has been implicated in the activation of RAF kinases, however, the mechanism and structural basis remain elusive. We developed a system to study KRAS dimerization on nanodiscs using paramagnetic relaxation enhancement (PRE) NMR spectroscopy, and determined distinct structures of membrane-anchored KRAS dimers in the active GTP- and inactive GDP-loaded states. Both dimerize through an α4–α5 interface, but the relative orientation of the protomers and their contacts differ substantially. Dimerization of KRAS-GTP, stabilized by electrostatic interactions between R135 and E168, favors an orientation on the membrane that promotes accessibility of the effector-binding site. Remarkably, “cross”-dimerization between GTP- and GDP-bound KRAS molecules is unfavorable. These models provide a platform to elucidate the structural basis of RAF activation by RAS and to develop inhibitors that can disrupt the KRAS dimerization. The methodology is applicable to many other farnesylated small GTPases.     

Synthesis,crystal structures,spectral, electrochemical and magnetic properties of di-µ-phenoxido-bridged dinuclear copper(II) complexes with N-salicylidene-2-hydroxybenzylamine derivatives: axial coordination effect of dimethyl sulphoxide molecule

The template reaction of salicylaldehyde and its substituted derivatives and 2-hydroxy-5-bromobenzylamine or 2-hydroxy-5-chlorobenzylamine with copper(II) acetate in dimethyl sulphoxide (dmso) afforded eight dinuclear Cu(II) complexes, [Cu₂(L)₂(dmso)₂] (H₂ L = N-salicylidene-2-hydroxy-5-bromobenzylamine, N-salicylidene-2-hydroxy-5-chlorobenzylamine and their 5-bromo, 5-nitro and 5-methyl-substituted salicylidene derivatives). These Cu(II) complexes were characterised by IR and UV-VIS-NIR spectroscopy, electric conductivity, cyclic voltammetry, and temperature dependence of magnetic susceptibilities (4.5–300 K). In the THF solution, the complexes are nonelectrolytes and exhibit a characteristic CT band due to phenoxido-bridging at 360–384 nm. In the cyclic voltammograms, an irreversible reduction process was observed at ?1.18–1.54 V vs Fc/Fc⁺. Single-crystal X-ray crystallography revealed that two Cu(II) ions were bridged by the two phenoxido-oxygen atoms of the two Schiff-base ligands with axial coordination of dmso molecules forming a square pyramid with a Cu-Cu distance of 3.0628(8)–3.0931(6) °A. In accordance with the crystal structures, the magnetic interaction between the two Cu(II) ions is relatively anti-ferromagnetic with ?2J value of 386–575 cm^?1. The axial coordination effect of the dmso molecule was discussed in relation to the correlation between the Cu-O-Cu angle and the ?2J value.     

Optimisation of the analysis of anti-influenza drugs in wastewater and surface water

We optimised the simultaneous analysis of four analytes, namely three anti-influenza drugs (oseltamivir (OS), zanamivir (ZAN) and amantadine (AMN)) and one metabolite of OS (oseltamivir carboxylate (OC)), in sewage treatment plant (STP) influent and effluent by verifying the types and conditions of solid phase extraction (SPE) appropriate for LC-MS/MS analysis. In summary, these target analytes were extracted from aqueous samples (30–50 mL) by using strong cation-exchange SPE cartridges (500 mg adsorbent) under acidic conditions (pH 3–4). After washing of the cartridges with acidified water (pH 3.0, 3 mL) and methanol (3 mL), the analytes were eluted with a mixed solvent (2 mL) of 10% (v/v) triethylamine in a 1:1 (v/v) mixture of acetone and water. Application of this technique to the target compounds should yield a comprehensive understanding of the occurrence and fate of anti-influenza drugs in the water environment.     

S‐((Phenylsulfonyl)difluoromethyl)thiophenium Salts: Carbon‐Selective Electrophilic Difluoromethylation of β‐Ketoesters, β‐Diketones,and Dicyanoalkylidenes

S‐((Phenylsulfonyl)difluoromethyl)thiophenium salts were designed and prepared by a triflic acid catalyzed intramolecular cyclization of ortho‐ethynyl aryldifluoromethyl sulfanes. The thiophenium salts were found to be efficient as electrophilic difluoromehtylating reagents for introduction of a CF₂H group to sp³‐hybridized carbon nucleophiles such as of β‐ketoesters and dicyanoalkylidenes. The (phenylsulfonyl)difluoromethyl group can be readily transformed into CF₂H under mild reaction conditions. Enantioselective electrophilic difluoromethylation was also achieved in the presence of bis(cinchona) alkaloids.     

Multidimensional umbrella sampling and replica‐exchange molecular dynamics simulations for structure prediction of transmembrane helix dimers

Structural information of a transmembrane (TM) helix dimer is useful in understanding molecular mechanisms of important biological phenomena such as signal transduction across the cell membrane. Here, we describe an umbrella sampling (US) scheme for predicting the structure of a TM helix dimer in implicit membrane using the interhelical crossing angle and the TM–TM relative rotation angles as the reaction coordinates. This scheme conducts an efficient conformational search on TM–TM contact interfaces, and its robustness is tested by predicting the structures of glycophorin A (GpA) and receptor tyrosine kinase EphA1 (EphA1) TM dimers. The nuclear magnetic resonance (NMR) structures of both proteins correspond to the global free‐energy minimum states in their free‐energy landscapes. In addition, using the landscape of GpA as a reference, we also examine the protocols of temperature replica‐exchange molecular dynamics (REMD) simulations for structure prediction of TM helix dimers in implicit membrane. A wide temperature range in REMD simulations, for example, 250–1000 K, is required to efficiently obtain a free‐energy landscape consistent with the US simulations. The interhelical crossing angle and the TM–TM relative rotation angles can be used as reaction coordinates in multidimensional US and be good measures for conformational sampling of REMD simulations. © 2013 Wiley Periodicals, Inc.     

Large-eddy simulation of turbulent channel flows with conservative IDO scheme

The resolution of a numerical scheme in both physical and Fourier spaces is one of the most important requirements to calculate turbulent flows. A conservative form of the interpolated differential operator (IDO-CF) scheme is a multi-moment Eulerian scheme in which point values and integrated average values are separately defined in one cell. Since the IDO-CF scheme using high-order interpolation functions is constructed with compact stencils, the boundary conditions are able to be treated as easy as the 2nd-order finite difference method (FDM). It is unique that the first-order spatial derivative of the point value is derived from the interpolation function with 4th-order accuracy and the volume averaged value is based on the exact finite volume formulation, so that the IDO-CF scheme has higher spectral resolution than conventional FDMs with 4th-order accuracy. The computational cost to calculate the first-order spatial derivative with non-uniform grid spacing is one-third of the 4th-order FDM. For a large-eddy simulation (LES), we use the coherent structure model (CSM) in which the model coefficient is locally obtained from a turbulent structure extracted from a second invariant of the velocity gradient tensor, and the model coefficient correctly satisfies asymptotic behaviors to walls.     

Two Distinct Structures of Membrane‐Associated Homodimers of GTP‐ and GDP‐Bound KRAS4B Revealed by Paramagnetic Relaxation Enhancement

KRAS homo‐dimerization has been implicated in the activation of RAF kinases, however, the mechanism and structural basis remain elusive. We developed a system to study KRAS dimerization on nanodiscs using paramagnetic relaxation enhancement (PRE) NMR spectroscopy, and determined distinct structures of membrane‐anchored KRAS dimers in the active GTP‐ and inactive GDP‐loaded states. Both dimerize through an α4–α5 interface, but the relative orientation of the protomers and their contacts differ substantially. Dimerization of KRAS‐GTP, stabilized by electrostatic interactions between R135 and E168, favors an orientation on the membrane that promotes accessibility of the effector‐binding site. Remarkably, “cross”‐dimerization between GTP‐ and GDP‐bound KRAS molecules is unfavorable. These models provide a platform to elucidate the structural basis of RAF activation by RAS and to develop inhibitors that can disrupt the KRAS dimerization. The methodology is applicable to many other farnesylated small GTPases.     

18π‐Electron Tautomeric Benziphthalocyanine: A Functional Near‐Infrared Dye with Tunable Aromaticity

Dihydroxybenziphthalocyanine 1 , with bulky aryloxy groups, has been synthesized and characterized by X‐ray crystallography, NMR and UV/Vis‐NIR spectroscopy, and theoretical calculations. Macrocycle 1 is the first example of an aromatic benziphthalocyanine with an 18π‐electron structure, and was found to exist as an equilibrium mixture of weakly aromatic and strongly aromatic tautomers. The aromaticity and near‐IR absorption can be controlled by chemical modification at the reactive resorcinol moiety and by variation of the solvent.     

Vertex‐disjoint cycles containing specified vertices in a bipartite graph

A minimum degree condition is given for a bipartite graph to contain a 2‐factor each component of which contains a previously specified vertex. © 2004 Wiley Periodicals, Inc. J Graph Theory 46: 145–166, 2004     

Excess resistance in the superconducting transition of a mesoscopic Al disk

We report resistance measurements on a mesoscopic Al disk whose size is comparable to the superconducting coherence length. As the magnetic field increases, resistance peaks successively appear and some of the peak resistances are larger than the normal state value R_N. These peaks are ascribed to the transitions between different vortex states in the superconducting Al disk. The experimental results suggest that some anomalous energy dissipation is caused by the dynamics of the vortices in the confined geometry.