Influence of the membrane potential on the protonation of bacteriorhodopsin: insights from electrostatic calculations into the regulation of proton pumping

Proton binding and release are elementary steps for the transfer of protons within proteins, which is a process that is crucial in biochemical catalysis and biological energy transduction. Local electric fields in proteins affect the proton binding energy compared to aqueous solution. In membrane proteins, also the membrane potential affects the local electrostatics and can thus be crucial for protein function. In this paper, we introduce a procedure to calculate the protonation probability of titratable sites of a membrane protein in the presence of a membrane potential. In the framework of continuum electrostatics, we use a modified Poisson-Boltzmann equation to include the influence of the membrane potential. Our method considers that in a transmembrane protein each titratable site is accessible for protons from only one side of the membrane depending on the hydrogen bond pattern of the protein. We show that the protonation of sites receiving their protons from different sides of the membrane is differently influenced by the membrane potential. In addition, the effect of the membrane potential is combined with the effect of the pH gradient resulting from proton pumping. Our method is applied to bacteriorhodopsin, a light-activated proton pump. We find that the proton pumping of this protein might be regulated by Asp115, a conserved residue for which no function has been identified yet. According to our calculations, the interaction of Asp115 with Asp85 leads to the protonation of the latter if the pH gradient or the membrane potential becomes too large. Since Asp85 is the primary proton acceptor in the photocycle, bacteriorhodopsin molecules in which Asp85 is protonated cannot pump protons. Furthermore, we estimate how the membrane potential affects the energetics of the individual proton-transfer reactions of the photocycle. Most reactions, except the initial proton transfer from the Schiff base to Asp85, are influenced. Our calculations give new insights into the mechanism with which bacteriorhodopsin senses the membrane potential and the pH gradient and how the proton pumping is regulated by these parameters.     

Pi-complex structure of gaseous benzene-NO cations assayed by IR multiple photon dissociation spectroscopy

[C(6)H(6)NO](+) ions, in two isomeric forms involved as key intermediates in the aromatic nitrosation reaction, have been produced in the gas phase and analyzed by IR multiple photon dissociation (IRMPD) spectroscopy in the 800-2200 cm(-)(1) fingerprint wavenumber range, exploiting the high fluence and wide tunability of a free electron laser (FEL) source. The IRMPD spectra were compared with the IR absorption spectra calculated for the optimized structures of potential isomers, thus allowing structural information on the absorbing species. [C(6)H(6)NO](+) ions were obtained by two routes, taking advantage of the FEL coupling to two different ion traps. In the first one, an FT-ICR mass spectrometer, a sequence of ion-molecule reactions was allowed to occur, ultimately leading to an NO(+) transfer process to benzene. The so-formed ions displayed IRMPD features characteristic of a [benzene,NO](+) pi-complex structure, including a prominent band at 1963 cm(-)(1), within the range for the N-O bond stretching vibration of NO (1876 cm(-)(1)) and NO(+) (2344 cm(-)(1)). A quite distinct species is formed by electrospray ionization (ESI) of a methanol solution of nitrosobenzene. The ions transferred and stored in a Paul ion trap showed the IRMPD features of substituent protonated nitrosobenzene, the most stable among conceivable [C(6)H(6)NO](+) isomers according to computations. It is noteworthy that IRMPD is successful in allowing a discrimination between isomeric [C(6)H(6)NO](+) species, whereas high-energy collision-induced dissociation fails in this task. The [benzene,NO](+) pi-complex is characterized by IRMPD spectroscopy as an exemplary noncovalent ionic adduct between two important biomolecular moieties.     

Intramolecular electron transfer in the photochemistry of some nitrophenyldihydropyridines

4-Phenyl-1,4-dihydropyridine-3,5-dicarboxylates contain two pi chromophores separated by an sp3 carbon. The lowest singlet is localized on the dihydropyridine moiety (1PyH2-Ph) and emits a blue fluorescence (with close to unitary efficiency in glass at 77 K). In 3-nitrophenyl derivatives (PyH2-PhNO2, some of which are photolabile drugs) the fluorescence is completely quenched. Reasonably, this is due to intramolecular electron transfer between the close-lying donor and acceptor moieties to give the charge-separated species (PyH2*+-PhNO2*-). In EPA glass at 77 K, back-electron transfer gives the dihydropyridine-localized triplet (3PyH2-PhNO2), which emits a yellow phosphorescence. In solution, deprotonation from the radical cation on the dihydropyridine moiety initiates rearomatization, finally giving Py-PhNO2 with low quantum yield (5 x 10(-4) to 5 x 10(-3), increasing up to 0.013 by irradiation at 254 nm, where direct excitation of the nitrophenyl chromophore contributes). In the presence of triethylamine, the reaction changes to neat reduction of the nitro group. When a tethered alkylamino group is present, oxidative degradation of that moiety occurs, again via an electron-transfer intramolecular process. This has been found with the drug nicardipine, where photodegration is more efficient (phi 0.02 to 0.1). Donor-acceptor dyads of this type, easily available through the Hantzsch synthesis, may be useful for building new photoinduced electron-transfer systems.     

Measurement of PAHs in environmental matrices: results from an interlaboratory comparison on the different steps of the measurement procedure

Proficiency testing (PT) is becoming a feature of laboratory accreditation and the PT results are used to assess the technical competence of the participating laboratories. ISPRA (former APAT) plays an important role in supporting the Italian laboratories belonging to the network of the Regional Environmental Agencies to improve the quality of their analytical measurements. As a consequence, ISPRA organized an interlaboratory comparison to assess the performance of the laboratories on PAH measurement procedure. The interlaboratory comparison was drawn separating the different steps of the measurement (from the extraction to the instrumental measurement). Two matrix reference materials: (1) a polluted soil and (2) an extract reference material of the same polluted soil and a “blind” PAH mixture standard stock solution were distributed to 59 Italian laboratories. The results of interlaboratory comparison showed a significant dispersion of the PAH measurements that masks the effects of the different extraction and cleanup procedures used, but it is consistent with the results of other European interlaboratory comparisons.     

Simultaneous determination of multibenzodiazepines by HPLC/UV: Investigation of liquid-liquid and solid-phase extractions in human plasma

A method for simultaneous determination of seven benzodiazepines (BZPs) (flunitrazepam, clonazepam, oxazepam, lorazepam, chlordiazepoxide, nordiazepam and diazepam using N-desalkylflurazepam as internal standard) in human plasma using liquid-liquid and solid-phase extractions followed by high-performance liquid chromatography (HPLC) is described. The analytes were separated employing a LC-18 DB column (250 mm × 4.6 mm, 5 μm) at 35 °C under isocratic conditions using 5 mM KH₂PO₄ buffer solution pH 6.0:methanol:diethyl ether (55:40:5, v/v/v) as mobile phase at a flow rate of 0.8 mL min⁻¹. UV detection was carried out at 245 nm. Employing LLE, the best conditions were achieved with double extraction of 0.5 mL plasma using ethyl acetate and Na₂HPO₄ pH 9.5 for pH adjusting. Employing SPE, the best conditions were achieved with 0.5 mL plasma plus 3 mL 0.1 M borate buffer pH 9.5, which were then passed through a C18 cartridge previously conditioned, washed for 3 times with these solvents: 3 mL 0.1 M borate buffer pH 9.5, 4 mL Milli-Q water and 1 mL acetonitrile 5%, finally the BZPs elution was carried with diethyl ether:n-hexane:methanol (50:30:20). In both methods the solvent was evaporated at 40 °C under nitrogen flow. The validation parameters obtained in LLE were linearity range of 50-1200 ng mL⁻¹ plasma (r ≥ 0.9927), limits of quantification of 50 ng mL⁻¹ plasma, within-day and between-day CV% and E% for precision and accuracy lower than 15%, and recovery above 65% for all BZPs. In SPE, the parameter obtained were linearity range of 30-1200 ng mL⁻¹ plasma (r ≥ 0.9900), limits of quantification of 30 ng mL⁻¹ plasma, within-day and between-day CV% and E% for precision and accuracy lower than 15% and recovery above 55% for all BZPs. These extracting procedures followed by HPLC analysis showed their suitable applicability in order to examine one or more BZPs in human plasma. Moreover, it could be suggested that these procedures might be employed in various analytical applications, in special for toxicological/forensic analysis.     

Formation of multifunctional ligands by nucleophilic addition of alcohols and thiols to the alkyne groups in compound C5H5FeC5H4CCSCCH: Reactivity studies

The multifunctional ligands [(Z)-FcCCSC(H)C(H)XR] [X = O, R = Me (2a); X = O, R = Et (2b); X = S, R = Ph (3); X = S, R = C₆F₅ (5)] and [(Z,Z)-Fc(SR)CC(H)SC(H)C(H)SR] [R = Ph (4), C₆F₅ (6)] have been prepared through hydroalkoxylation and hydrothiolation processes of the alkyne groups in the compound FcCCSCCH 1. Reactions between compound 3 and the carbonyl metals Co₂(CO)₈, Os₃(CO)₁₀(NCMe)₂ and Fe₂(CO)₉ have allowed the synthesis of the polynuclear compounds [(Z)-{Co₂(CO)₆}(μ-η²-FcCCSC(H)C(H)SPh)] 9, [(Z)-Os₃(CO)₉(μ-CO){μ₃-η²-FcCCSC(H)C(H)(SPh)}] 10 and [(Z)-{Fe₃(CO)₉}[μ₃,η³-(CCS)-FcCCSC(H)C(H)(SPh)] 11. All the compounds have been characterized by elemental analysis, ¹H and ¹³C{¹H} NMR spectroscopy, mass spectrometry and the crystal structure of compounds [(Z)-FcCCSC(H)C(H)OMe] 2a and [{Co₂(CO)₆}₂(μ-η²:η²-FcCCSCCSiMe₃)] 7 have been solved by X ray diffraction analysis.     

Functional isocyanide metal complexes as building blocks for supramolecular materials: hydrogen-bonded liquid crystals

Gold, palladium and platinum complexes with an unusual isocyanide ligand containing a carboxylic acid function, [AuCl(CNC(6)H(4)COOH)], cis-[MI(2)(CNC(6)H(4)COOH)(2)] and trans-[MI(2)(CNC(6)H(4)COOH)(2)] (M = Pd, Pt) have been isolated. The carboxylic acid group of the coordinated isocyanide acts as a hydrogen donor for hydrogen-bonding and three series of stable hydrogen-bonded liquid crystalline metal complexes have been prepared with decyloxystilbazole. Although all the metal acid derivatives used are not mesomorphic, and decyloxystilbazole only shows an ordered Smectic E phase, four out of the five hydrogen-bonded decyloxystilbazole complexes studied display enantiotropic smectic A or nematic mesophases. The single crystal X-ray diffraction structure of trans-[PdI(2)(CNC(6)H(4)COOH)(2)].C(4)H(8)O(2) has been determined and confirms the formation of a supramolecular array in the solid state supported by hydrogen-bonding.     

Biexcitons in artificial molecules with in-plane magnetic field

We theoretically investigate the effect of a magnetic field perpendicular to the tunneling direction on the ground-state properties of biexcitons in coupled quantum dots. The single-particle states are computed by numerically solving the 3D Scrödinger equation. The biexciton states are obtained by means of a configuration-interaction approach, which fully accounts for the intra- and inter-dot Coulomb correlations. We show that the biexciton ground state undergoes non-trivial transitions as a function of the applied magnetic field, which can be traced back to unexpected carrier localizations.     

Tuning the structural and magnetic properties of thermally robust coordination polymers

Thermally robust materials of the M(5-X-pyrimidin-2-olate)2 type [M = Co, X = Cl (1(Cl)), X = Br (1(Br)), X = I (1(I)); M = Zn, X = Cl (2(Cl)), X = Br (2(Br)), X = I (2(I))] have been synthesized. Their X-ray powder diffraction structural characterization has revealed that they crystallize as I2d diamondoid frameworks, isomorphous to those of the pristine [M(pyrimidin-2-olate)2]n analogues (1(H), M = Co; 2(H), M = Zn). The magnetic measurements of the 1(X) series at magnetic fields of 100, 300, and 5000 Oe reveal a weak ferromagnetic ordering taking place below the Néel temperature (T(N) approximately 20 K), arising from spin canting phenomena of the antiferromagnetically coupled cobalt centers. Moreover, magnetic hysteresis studies carried out on the 1(X) series at 2 K reveal a strong dependence of both the coercive field H(coer) (2500, 1000, 775, and 500 Oe for 1(Br), 1(Cl), 1(I), and 1(H), respectively) and the remnant magnetization M(rem) (0.0501 mu(B) for 1(Br) and 1(Cl), 0.0457 mu(B) for 1(I), and 0.0358 mu(B) for 1(H)) on the 5-substituent of the pyrimidin-2-olates. The molecular alloys [Co(5-Y-pyrimidin-2-olate)2] (Y = Cl/Br, 1(Cl/Br)) and [Co(5-Y'-pyrimidin-2-olate)2] (Y' = Br/I, 1(Br/I)) have also been prepared and characterized, proving that they have intermediate properties. These materials combine interesting functional properties, such as chemical inertness, magnetism, photoluminescence, and (although weak) SHG activity.