Elongated Bacterial Pili as a Versatile Alignment Medium for NMR Spectroscopy

In NMR spectroscopy, residual dipolar couplings (RDCs) have emerged as one of the most exquisite probes of biological structure and dynamics. The measurement of RDCs relies on the partial alignment of the molecule of interest, for example by using a liquid crystal as a solvent. Here, we establish bacterial type 1 pili as an alternative liquid-crystalline alignment medium for the measurement of RDCs. To achieve alignment at pilus concentrations that allow for efficient NMR sample preparation, we elongated wild-type pili by recombinant overproduction of the main structural pilus subunit. Building on the extraordinary stability of type 1 pili against spontaneous dissociation and unfolding, we show that the medium is compatible with challenging experimental conditions such as high temperature, the presence of detergents, organic solvents or very acidic pH, setting it apart from most established alignment media. Using human ubiquitin, HIV-1 TAR RNA and camphor as spectroscopic probes, we demonstrate the applicability of the medium for the determination of RDCs of proteins, nucleic acids and small molecules. Our results show that type 1 pili represent a very useful alternative to existing alignment media and may readily assist the characterization of molecular structure and dynamics by NMR.     

Novel adsorbent based on silkworm chrysalides for removal of heavy metals from wastewaters

In this contribution, maximum capacity for adsorption of Pb(2+), Ni(2+), and Cu(2+) by silkworm chrysalides (SC) was determined. The raw silkworm chrysalides (SC(r)) and chrysalides after acidic washing (SC(w)) were used. Chitin (CT), extracted from SC, and chitosan (CS), with 85% deacetylation, were employed as reference samples. Adsorption tests showed that all the studied adsorbents exhibited excellent performance in removal of metals. The choice of a more appropriate adsorbent is related to its efficiency for removal of a specific metal. The studied materials presented different intensities for metal adsorption as follows: (i) Ni(2+)>Cu(2+)>Pb(2+) for SC(r); (ii) Pb(2+)>Cu(2+)>Ni(2+) for SC(w); (iii) Ni(2+)>Cu(2+)>Pb(2+) for CT; and (iv) Cu(2+)>Pb(2+)>Ni(2+) for CS. Metal adsorption onto SC(r) and CS was analyzed by Freundlich and Langmuir isotherm equations. Adsorption values for CS-Pb and SC(r)-Ni were provided by the Freundlich model, while the adsorption values for CS-Cu, CS-Ni, SC(r)-Pb, and SC(r)-Cu were provided by the Langmuir model. The studied adsorbents are suitable for use in treatment of wastewater. From the economic point of view, the use of SC(r) as an adsorbent of heavy metals (mainly Ni(2+)) on the large industrial scale would be more appropriate.     

trans-cis Photoisomerization of the styrylpyridine Ligand in [Re(CO)3(2,2'-bipyridine)(t-4-styrylpyridine)]+: role of the metal-to-ligand charge-transfer excited states

The trans-cis isomerization of the styrylpyridine carbon-carbon double bond induced by visible light irradiation in fac-[Re(CO)(3)(bpy)(stpy)](+) (bpy = 2,2'-bipyridine; stpy = t-4-styrylpyridine) has been investigated by means of quantum-chemical methods. The structures of the various cis and trans conformers of [Re(CO)(3)(bpy)(stpy)](+) have been optimized at the density functional theory (DFT) level. Three rotational conformers for the most stable trans isomer lie within 2.3 kJ mol(-1) each other. The energy difference between the cis and trans isomers is 27.0 kJ mol(-1). The electronic spectroscopy of the most stable conformers has been investigated by time-dependent DFT (TD-DFT) and complete active space self-consistent field/CAS second order perturbation theory (CASSCF/CASPT2) calculations. The lowest absorption bands are dominated by metal-to-ligand charge-transfer (MLCT, d(Re)-->pi*(bpy)) transitions calculated at about 25,000 cm(-1) and by a strong intraligand (1)IL (pi(stpy)-->pi*(stpy)) transition in the near UV region. On the basis of CASSCF potential energy curves (PECs) calculated as a function of the torsion angle of the C=C bond of the styrylpyridine ligand, it is shown that the role of the low-lying MLCT states is important in the photoisomerization mechanism. In contrast to the free organic ligand, in which the singlet mechanism is operational via the (1)IL (S(1)) and electronic ground (S(0)) states, coordination to the rhenium steers the isomerization to the triplet PEC corresponding to the (3)IL state. From the (3)IL(t) (t = trans) the system evolves to the perpendicular intermediate (3)IL(p) (p = perpendicular) following a 90 degrees rotation around the styrylpyridine C=C bond. The metal center acts as a photosensitizer because of the presence of photoactive MLCT states under visible irradiation. The position of the crossing between the (3)IL and electronic ground state PEC determines the quantum yield of the isomerization process.     

Electron density redistribution accounts for half the cooperativity of alpha helix formation

The energy of alpha helix formation is well known to be highly cooperative, but the origin and relative importance of the contributions to helical cooperativity have been unclear. Here we separate the energy of helix formation into short range and long range components by using two series of helical dimers of variable length. In one dimer series two monomeric helices interact by forming hydrogen bonds, while in the other they are coupled only through long range, primarily electrostatic interactions. Using Density Functional Theory, we find that approximately half of the cooperativity of helix formation is due to electrostatic interactions between residues, while the other half is due to nonadditive many-body effects brought about by redistribution of electron density with helix length.     

Copper acetylacetonate anchored onto amine-functionalised clays

Copper (II) acetylacetonate was immobilised directly onto two clays, laponite (Lap) and K10-montmorillonite (K10), and after their amine functionalisation with (3-aminopropyl)triethoxysilane (APTES). All the materials were characterised by nitrogen adsorption isotherms at -196 degrees C, elemental analysis, TG-DSC, XRD, and IR spectroscopy. The K10-based materials were also characterised by XPS. The APTES-functionalised K10 showed higher copper loading than K10, indicating that the clay functionalisation enhanced the complex immobilisation; on the contrary, in Lap-based materials higher metal content was obtained by direct complex anchoring, probably due to the delaminated nature of Lap which induced the particles aggregation on functionalisation with APTES. All the results pointed out that the Cu complex was anchored onto the amine-functionalised clays by Schiff condensation between the amine groups of anchored APTES and the carbonyl groups of the acetylacetonate ligand, whereas direct immobilisation proceeded mostly through interaction between the metal centre and the clay surface hydroxyl groups.     

Voltage-dependent anion channel transports calcium ions through biomimetic membranes

The mitochondrial outer membrane channel (VDAC), a central player in mitochondria and cell death, was reconstituted in polymer-supported phospholipid bilayers. Highly purified VDAC was first reconstituted in vesicles; channel properties and NADH-ferricyanide reductase activity were ascertained before deposition onto solid substrates. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-N-hydroxysuccinimide mixed vesicles containing VDAC were linked onto amine-grafted surfaces (glass and gold) and disrupted to form a VDAC-containing polymer-tethered planar bilayer. Surface plasmon spectroscopy, fluorescence microscopy, and atomic force microscopy measurements ascertained the membrane thickness, fluidity, and continuity. VDAC reconstituted in bilayers efficiently transported calcium ions and was modulable by two channel blockers, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and l-glutamate. The novel setup may allow the study of the assembly of a polyprotein complex centered on VDAC and its role in mitochondrial biology, calcium fluxes, and apoptosis.     

Synthesis of a Naphthalimide Functionalized Calix[4]arene; A Host Type Fluorophore for Inclusion Compounds in Organic Medium

This article reports the synthesis and the properties of 5,11,17,23-tetra(t-butyl)-25,27-bis-(ethoxy-1,8-naphthalimide)-26,28-hidroxy-calix[4]arene and the formation of an inclusion compound in organic medium. This functionalized calix[4]arene was conceived as the association of a potential host species with a good fluorophore for optical sensoring purposes. Calix[4]-NI as we will call it, maintains its ‘cone-pinched’ configuration and exhibits typical naphthalimide fluorescence bands in non-polar solvents. Its ability to interact with guest species via hydrogen bonding in its endo-hydrophilic cavity to form inclusion compounds was verified with absorption and fluorescence measurements using N-ethanol-1,8-naphthalimide as guest species, which was projected to fit exactly the host cavity and to interact with its naphthalimide π electrons. For this reason, it was possible to follow the formation of the inclusion compound with electronic spectroscopy.     

Analysis of the essential oil from aerial parts of Eupatorium cannabinum subsp. corsicum (L.) by gas chromatography with electron impact and chemical ionization mass spectrometry

Eupatorium cannabinum subsp. corsicum (L.), an aromatic plant, is an endemic subspecies from Corsica. The essential oil from aerial parts of E. cannabinum subsp. corsicum was studied by GC, GC/MS and 13C NMR. One hundred and forty-seven components were identified representing 93.6% of the total amount. The main constituents are germacrene D (28.5%), alpha-phellandrene (19.0%) and para-cymene (5.2%). A particularity of this essential oil is the presence of monoterpene esters derived from nerol, lavandulol, borneol, thymol and 8,9-dehydrothymol. These compounds have been investigated using GC/MS in different ionization modes like electron impact (EI), positive chemical ionization (PCI) and negative chemical ionization (NCI).     

Enhanced fluorescence from arrays of nanoholes in a gold film

Arrays of sub-wavelength holes (nanoholes) in gold films were used as a substrate for enhanced fluorescence spectroscopy. Seven arrays of nanoholes with distinct periodicities (distances between the holes) were fabricated. The arrays were then spin-coated with polystyrene films containing different concentrations of the fluorescent dye oxazine 720. The dye was excited via resonant extraordinary transmission of the laser source through the nanoholes. Enhanced fluorescence was observed when the geometric characteristics of the arrays allowed for an enhancement in the transmitted excitation. This enhancement occurred via surface plasmon excitation by the laser and a consequential increase in the local electromagnetic field in a sub-wavelength region at the metal-film interface. It was demonstrated that the sensitivity of the fluorescence measurement (change in signal vs change in dye concentration in the polymer film) is significantly larger at the surface plasmon resonance conditions than that obtained from equivalent films on glass substrates. Enhancement factors for the fluorescence emission were calculated for each array, with a maximum enhancement of close to 2 orders of magnitude as compared to the emission of films on glass. The results presented here indicate that arrays of nanoholes are interesting substrates for the development of fluorescence sensors based on surface plasmon resonance, as they provide a platform that allows both spatial confinement and enhancement of excitation light. Moreover, the collinear characteristics of the present optical setup, due to the resonant extraordinary transmission through the nanohole arrays, are more conducive to miniaturization and chip integration than more traditional experimental geometries.