Understanding the HIV-1 protease reactivity with DFT: what do we gain from recent functionals?

The modeling of HIV-1 plays a crucial role in the understanding of its reactivity and its interactions with specific drugs. In this work, we propose a medium sized model to test the ability of a variety of quantum chemistry approaches to provide reasonable geometric parameters and energetics for this system. Although our model is large enough to include the main polarizing groups of the active site, it is small enough to be used within full quantum studies up to the second order Møller-Plesset (MP2) level with extrapolations to coupled cluster CCSD(T) level. These high level calculations are used as reference to assess the ability of electronic structure methods (semiempirical and DFT) to provide accurate geometries and energies for the HIV-1 protease reaction. All semiempirical methods fail to describe the geometry of the protease active site. Within DFT, pure generalized gradient approximation (GGA) functionals have difficulty in reproducing the reaction energy and underestimate the barrier. Hybrid and/or meta GGA approaches do not yield a consistent improvement. The best results are obtained with hybrid GGA B3LYP or X3LYP and with hybrid meta GGA functionals with a fraction of exact exchange around 30-40%, such as M06, B1B95, or BMK functionals. On the basis of these results, we propose an accurate and computationally efficient strategy, employing quantum chemistry methods. This is applied here to study the protonation state of the reaction intermediate and could be easily used in further QM/MM studies.

Dynamical studies of the ozone isotope effect: A status report.

Dynamical studies of the recombination of O and O2 to form ozone are reviewed. The focus is the intriguing isotope dependence of the recombination rate coefficient as observed by Mauersberger and coworkers in the last decade. The key quantity for understanding of this dependence appears to be the difference of zero-point energies of the two fragmentation channels to which excited ozone can dissociate, i.e., X + YZ <-- XYZ* --> XY + Z, where X, Y, and Z stand for the three isotopes of oxygen. Besides the isotope dependence, the variation of the recombination rate coefficient with pressure and temperature is also addressed. Despite the numerous approaches of recent years, the recombination of ozone is far from being satisfactorily explained; there are still several essential questions to be solved by detailed theoretical analysis. We mainly discuss--and critically assess--the results of our own investigations of the ozone kinetics. The work of other research groups is also evaluated.

Structural changes induced by NaCl in companion and transfer cells of Medicago sativa blades.

Medicago sativa var. Gabes is a perennial glycophyte that develops new shoots even in high salinity (150 mM NaCl). In the upper exporting leaves, K(+) is high and Na(+) is low by comparison with the lower leaves, where Na(+) accumulation induces chlorosis after 4 weeks of NaCl treatment. By secondary ion mass spectroscopy, a low Na(+)/K(+) ratio was detected in the phloem complex of blade veins in these lower leaves. By transmission electron microscopy, the ultrastructural features were observed in the phloem complex. In the upper leaves of both control and NaCl-treated plants, companion cells in minor veins were found to be transfer cells. These cells may well be involved in the intravenous recycling of ions and in Na(+) flowing out of exporting leaves. Under the effect of NaCl, companion cells in the main veins develop transfer cell features, which may favor the rate of assimilate transport from exporting leaves toward meristems, allowing the positive balance necessary for the survival in salt conditions. These features no longer assist the lower leaves when transfer cells are necrotized in both minor and main veins of NaCl-treated plants. As transfer cells are the only degenerating phloem constituent, our observations emphasize their role in controlling nutrient (in particular, Na(+)) fluxes associated with the stress response.

The presence of Lewis a epitopes in Arabidopsis thaliana glycoconjugates depends on an active alpha4-fucosyltransferase gene.

The presence of an alpha4-fucosyltransferase in plants was first deduced from the characterization of Lewis-a glycoepitopes in some N-glycans. The first plant gene encoding an alpha4-fucosyltransferase was recently cloned in Beta vulgaris. In the present paper we provide evidence for the presence of an alpha4-fucosyltransferase in A. thaliana by measurement of this glycosyltransferase activity from a purified microsomal preparation and by immunolocalization of Le(a) epitopes on glycans N-linked to glycoproteins located to the Golgi apparatus and on the cell surface. The corresponding gene AtFT4 (AY026941) was characterized. A unique copy of this gene was found in A. thaliana genome, and a single AtFT4 transcript was revealed in leaves, in roots, and at a lower extent in flowers. The coding sequence of AtFT4 gene is interrupted by two introns spanning 465 bp and 84 bp, respectively. The putative 393-amino-acid protein (44 kDa, pI: 6.59) contains an N-terminal hydrophobic region and one potential N-glycosylation site, but AtFT4 has poor homology (less than 30%) to the other alpha3/4-fucosyltransferases except for motif II. When expressed in COS 7 cells the protein is able to transfer Fuc from GDP-Fuc to a type 1 acceptor substrate, but this transferase activity is detected only in the culture medium of transfected cells

Structure of triamidoaluminum complexes: a theoretical ab initio/IMOMM study.

The electronic structure of three-coordinated complexes of aluminum Al(NR2)3 has been studied through theoretical calculations. In the unsubstituted system Al(NH2)3, ab initio calculations (MP2/6-31G(d,p) level) show that the optimal geometry results from a conrotatory motion of the three amido substituents starting from the fully conjugated planar species. The energy difference between these two structures is found to be small (less than 0.5 kcal/mol). In methylated species Al(NMe2)3, steric effects become important and the planar geometry is destabilized. The conrotatory geometry corresponds to the absolute minimum and is located 14.2 kcal/mol below the planar structure (MP2/6-31G(d,p) level). Several coupled motions of the amido substituents have been computed and have been found to stabilize the system with respect to the fully conjugated structure. A rough estimate of the steric repulsion is calculated by comparison between the unsubstituted and methylated species. Finally, the real molecules Al[N(SiMe3)2]3 and Al(NiPr2)3 have been studied through a coupled quantum mechanics/molecular mechanics method. In accordance with the experimental data, it is found that the conrotatory minimum is the absolute minimum in the R = SiMe3 case whereas a less symmetrical minimum is found in the R = iPr case. In this last minimum, an amido group is almost deconjugated and the two other groups move in a conrotatory manner. The different behavior of these two systems may originate from the quasi-spherical shape of the SiMe3 group, which leads to unavoidable steric repulsion.

Contrasting behavior of tetracene and perylene in collision-induced dissociation: a theoretical interpretation.

Dimethyl ether chemical ionization mass spectrometry of polycyclic aromatic hydrocarbons (PAHs) leads to [M + 13](+) and [M + 45](+) ions. The process leading to these ions is sensitive to the proton affinity of the PAH. Collision-induced dissociation observations on [M + 45](+) ion also show that tetracene has a peculiar reactivity in comparison with perylene, despite the similar physico-chemical properties of these two molecules. Ab initio calculations were used to establish a potential energy profile for the mechanistic pathway of [M + 13](+) and [M + 45](+) formation. [M + 45](+) ions result from the addition of CH(3)-O-CH(2)(+) to PAHs. A 1,2-hydride transfer followed by a 1,4-proton transfer and a loss of methanol subsequently lead to the formation of [M + 13](+) ions. For tetracene, the 1,2-hydride transfer does not occur, as it would lead to a thermodynamically unstable non-planar ion.

Increased Expression of Vacuolar Aquaporin and H+-ATPase Related to Motor Cell Function in Mimosa pudica L.

Mature motor cells of Mimosa pudica that exhibit large and rapid turgor variations in response to external stimuli are characterized by two distinct types of vacuoles, one containing large amounts of tannins (tannin vacuole) and one without tannins (colloidal or aqueous vacuole). In these highly specialized cells we measured the abundance of two tonoplast proteins, a putative water-channel protein (aquaporin belonging to the [gamma]-TIPs [tonoplast intrinsic proteins]) and the catalytic A-subunit of H+-ATPase, using either high-pressure freezing or chemical fixation and immunolocalization. [gamma]-TIP aquaporin was detected almost exclusively in the tonoplast of the colloidal vacuole, and the H+-ATPase was also mainly localized in the membrane of the same vacuole. Cortex cells of young pulvini cannot change shape rapidly. Development of the pulvinus into a motor organ was accompanied by a more than 3-fold increase per length unit of membrane in the abundance of both aquaporin and H+-ATPase cross-reacting protein. These results indicate that facilitated water fluxes across the vacuolar membrane and energization of the vacuole play a central role in these motor cells.

Distribution and Activity of the Plasma Membrane H+-ATPase in Mimosa pudica L. in Relation to Ionic Fluxes and Leaf Movements.

Plasma membrane H+-ATPase was immunolocalized in several cell types of the sensitive plant Mimosa pudica L., and transmembrane potentials were measured on cortical cells. In comparison with the nonspecialized cortical cells of the petiole or stem, the proton pump was highly expressed in motor cells. These immunological data are in close agreement with electrophysiological data, because the active component of the transmembrane potential was low in the nonspecialized cortical cells and high in motor cells. Therefore, motor cells contain the plasma membrane H+-ATPase required to mediate the ionic fluxes that are involved in circadian leaf movements and that are necessary to recover the turgor potential that is considerably affected by the large K+ and Cl- efflux associated with seismonastic movement. With the exception of sieve tubes, the phloem also had a high density of H+-ATPase. This suggests that the recovery of the transmembrane ionic gradients (K+ and Cl-), which is affected by various stimuli, is more energized by the companion and parenchyma cells than by the sieve elements. In addition, at the phloem/cortex interface collocytes displayed the required properties for lateral transduction of the action potential toward the pulvinal motor cells.

Control of Vascular Sap pH by the Vessel-Associated Cells in Woody Species (Physiological and Immunological Studies).

In Robinia wood, the vessel-associated cells form a continuous sleeve around the vessels. Variations in pH of the solution perfused through the vessels during the annual cycle and the opposing effects of carbonyl cyanide-m-chlorophenylhydrazone and fusicoccin on this pH value indicate that some living cells of the wood are involved in the control of vascular sap pH and that this control fluctuates with the seasons. The immunolocalization of the plasma membrane HT+-ATPase in Robinia wood was studied by the immunogold-silver-staining technique using an antibody raised against a conserved stretch of the cytoplasmic domain of the H+-ATPase. The immunostaining is much stronger in vessel-associated cells than in other living cell types (ray and axial parenchyma elements) of the secondary xylem. Our data show an efficient involvement of this cell type in the control of vascular sap pH.

Immunolocalization of the Plasma Membrane H+ -ATPase in Minor Veins of Vicia faba in Relation to Phloem Loading.

The immunolocalization of the plasma membrane H+ -ATPase, which generates a proton motive force energizing the uptake of inorganic and organic solutes, was studied by electron microscopy. The cells studied were in minor veins of Vicia faba L. exporting leaves, where photosynthates are supposed to be absorbed from the apoplast by phloem transfer cells. Immunologically detectable H+ -ATPase varied among the different cell types and was considerably denser in the transfer cells than in the other cell types, particularly in the sieve tube. Moreover, the distribution of the H+ -ATPase was not homogeneous in transfer cells, that pump being more concentrated in the region adjacent to the bundle sheath, phloem parenchyma, and xylem vessels than along the smooth part of the wall bordering the sieve tube. These results show that the plasma membrane infoldings of transfer cells possess the proton-pumping machinery required to energize an efficient uptake of photosynthates from the phloem apoplast and an efficient retrieval of nitrogenous compounds from the vascular sap.

Effects of Compounds Affecting Calcium Channels on Phytochrome- and Blue Pigment-Mediated Pulvinar Movements of Cassia fasciculata.

Verapamil and nifedipine, known as calcium channel blockers, inhibited the phytochrome-mediated movements induced on Cassia fasciculata leaflets by a light-off signal, whereas they had no effect on the ;blue' pigment-mediated movements induced by a light-on signal. LaCl(3) inhibited both types of reactions, but the inhibition of light-induced opening needed a 10 times higher concentration than that of dark-induced closure. Bay K 8644, an activator of calcium channels, increased the rate of dark-induced closure, whereas it had no effect on the light-induced opening. These data suggest that calcium ions are not mobilized in the same way in the two types of movements: possibly from external stores in the phytochrome-mediated reaction and from internal stores in the ;blue' pigment-mediated reaction.

Redistribution of potassium, chloride and calcium during the gravitropically induced movement of Mimosa pudica pulvinus.

When the leaves of Mimosa pudica are changed from their normal position in the gravitational field, they perform reversible compensatory movements by means of pulvini. These movements are not the result of growth processes but involve reversible turgor variations. These variation are concomitant with ion migrations within pulvini: during the gravitropic movement, K(+) and Cl(-) shift towards the adaxial half of the motor organ whereas Ca(2+) shifts towards the abaxial half. Compounds known to affect K(+) transport, tetraethylammonium chloride and valinomycin, do not hinder the gravitropic movement but inhibit strongly the seismonastic reaction. The same general result is obtained with compounds affecting anion transport, disulfonic stilbenes and 9-anthracene carboxylic acid. Calcium chelators inhibit the gravitropic movement more efficiently than the seismonastic reaction and the calcium ionophore A 23 187 increases both movements. The data obtained with these various compounds indicate that ions do not have the same functional importance in the regulation of the two different pulvinar movements.