Hybrid MC/QC simulations of water-assisted proton transfer in nucleosides. Guanosine and its analog acyclovir

To provide an in-depth insight into the molecular basis of spontaneous tautomerism in DNA and RNA base pairs, a hybrid Monte Carlo (MC)–quantum chemical (QC) methodology is implemented to map two-dimensional potential energy surfaces along the reaction coordinates of solvent-assisted proton transfer processes in guanosine and its analog acyclovir in aqueous solution. The solvent effects were simulated by explicit inclusion of water molecules that model the relevant part of the first hydration shell around the solute. The position of these water molecules was estimated by carrying out a classical Metropolis Monte Carlo simulation of dilute water solutions of the guanosine (Gs) and acyclovir (ACV) and subsequently analyzing solute–solvent intermolecular interactions in the statistically-independent MC-generated configurations. The solvent-assisted proton transfer processes were further investigated using two different ab initio MP2 quantum chemical approaches. In the first one, potential energy surfaces of the ‘bare’ finite solute–solvent clusters containing Gs/ACV and four water molecules (MP2/6-31+G(d,p) level) were explored, while within the second approach, these clusters were embedded in ‘bulk’ solvent treated as polarizable continuum (C-PCM/MP2/6-31+G(d,p) level of theory). It was found that in the gas phase and in water solution, the most stable tautomer for guanosine and acyclovir is the 1H-2-amino-6-oxo form followed by the 2-amino-6-(sZ)-hydroxy form. The energy barriers of the water-assisted proton transfer reaction in guanosine and in acyclovir are found to be very similar – 11.74 kcal mol^?1 for guanosine and 11.16 kcal mol^?1 for acyclovir, and the respective rate constants (k = 1.5?×?10¹ s^?1, guanosine and k = 4.09?×?10¹ s^?1, acyclovir), are sufficiently large to generate the 2-amino-6-(sZ)-hydroxy tautomer. The analysis of the reaction profiles in both compounds shows that the proton transfer processes occur through the asynchronous concerted mechanism.     

Factors affecting cation-anion uptake balance and iron acquisition in peanut plants grown on calcareous soils

Dicotyledonous plants subjected to Fe-deficiency stress can decrease pH in the rhizosphere by proton excretion and reduce ferric iron by an activated reduction system in the plasma membranes of the root or by reductants released from the roots. The efficiency by which these plants take up Fe may strongly depend on their cation-anion balance. This study presents results of two experiments conducted to evaluate the effect of K, growth stage and cultivar on ionic balance and Fe acquisition of peanut (Arachis hypogaea L.) plants.Potassium applications to the high calcareous soil (30.3% CaCO₃) favoured proton release, but did not ameliorate plant Fe acquisition. At the earliest stages of plant growth, anion uptake exceeded cation uptake due to intensive N uptake. With time, a shift in the ionic balance was observed as a result of predominant cation uptake. It appears that the relationship between H/OH-ion release and Fe nutrition of peanut plants is actually a complex phenomenon under soil conditions and depends on some soil parameters, such as CaCO₃ content. Even by enhanced H-ion release Fe nutrition of plants can be impaired if soil CaCO₃ is too high.     

The relationship between amide proton chemical shifts and secondary structure in proteins

Summary The parameters for HN chemical shift calculations of proteins have been determined using data from high-resolution crystal structures of 15 proteins. Employing these chemical shift calculations for HN protons, the observed secondary structure chemical shift trends of HN protons, i.e., upfield shifts on helix formation and downfield shifts on -sheet formation, are discussed. Our calculations suggest that the main reason for the difference in NH chemical shifts in helices and sheets is not an effect from the directly hydrogen-bonded carbonyl, which gives rise to downfield shifts in both cases, but arises from an additional upfield shift predicted in helices and originating in residues i-2 and i-3. The calculations also explain the well-known relationship between amide proton shifts and hydrogen-bond lengths. In addition, the HN chemical shifts of the distorted amphipathic helices of the GCN4 leucine zipper are calculated and used to characterise the solution structure of the helices. By comparing the calculated and experimental shifts, it is shown that in general the agreement is good between residues 15 and 28. The most interesting observation is that in the N-terminal half of the zipper, although both calculated and experimental shifts show clear periodicity, they are no longer in phase. This suggests that for the N-terminal half, in the true average solution structure the period of the helix coil is longer by roughly one residue compared to the NMR structures.     

pH-dependent cargo sorting from the Golgi

The vacuolar proton-translocating ATPase (V-ATPase) plays a major role in organelle acidification and works together with other ion transporters to maintain pH homeostasis in eukaryotic cells. We analyzed a requirement for V-ATPase activity in protein trafficking in the yeast secretory pathway. Deficiency of V-ATPase activity caused by subunit deletion or glucose deprivation results in missorting of newly synthesized plasma membrane proteins Pma1 and Can1 directly from the Golgi to the vacuole. Vacuolar mislocalization of Pma1 is dependent on Gga adaptors although no Pma1 ubiquitination was detected. Proper cell surface targeting of Pma1 was rescued in V-ATPase-deficient cells by increasing the pH of the medium, suggesting that missorting is the result of aberrant cytosolic pH. In addition to mislocalization of the plasma membrane proteins, Golgi membrane proteins Kex2 and Vrg4 are also missorted to the vacuole upon loss of V-ATPase activity. Because the missorted cargos have distinct trafficking routes, we suggest a pH dependence for multiple cargo sorting events at the Golgi.     

Cholesterol trafficking and raft-like membrane domain composition mediate scavenger receptor class B type 1-dependent lipid sensing in intestinal epithelial cells

Scavenger receptor Class B type 1 (SR-B1) is a lipid transporter and sensor. In intestinal epithelial cells, SR-B1-dependent lipid sensing is associated with SR-B1 recruitment in raft-like/ detergent-resistant membrane domains and interaction of its C-terminal transmembrane domain with plasma membrane cholesterol. To clarify the initiating events occurring during lipid sensing by SR-B1, we analyzed cholesterol trafficking and raft-like domain composition in intestinal epithelial cells expressing wild-type SR-B1 or the mutated form SR-B1-Q445A, defective in membrane cholesterol binding and signal initiation. These features of SR-B1 were found to influence both apical cholesterol efflux and intracellular cholesterol trafficking from plasma membrane to lipid droplets, and the lipid composition of raft-like domains. Lipidomic analysis revealed likely participation of d18:0/16:0 sphingomyelin and 16:0/0:0 lysophosphatidylethanolamine in lipid sensing by SR-B1. Proteomic analysis identified proteins, whose abundance changed in raft-like domains during lipid sensing, and these included molecules linked to lipid raft dynamics and signal transduction. These findings provide new insights into the role of SR-B1 in cellular cholesterol homeostasis and suggest molecular links between SR-B1-dependent lipid sensing and cell cholesterol and lipid droplet dynamics.     

No direct linkage between proton pumping and photosynthate unloading from seed coats of Phaseolus vulgaris L.

The energization of the active sucrose release from bean seed-coat halves was investigated. For this purpose, seed coat tissues adjacent to the apoplastic space were exposed to a variety of treatments and proton and photosynthate release were measured. Fusicoccin (10^–5 moll^–1) stimulated proton pump activities. Orthovanadate (2×10^–4 moll^–1) and abscisic acid (10^–5 moll^–1) diminished the proton extrusion evoked by fusicoccin. Fusicoccin inhibited sucrose release, whereas orthovanadate and abscisic acid stimulated it. Addition of 100 mmoll^–1 K⁺ had a promotory effect on photosynthate unloading, fading away with time. This extra unloading was linearly related to an enhanced proton loss. It was concluded that the photosynthate unloading apparently is not a proton/sucrose antiport and that a pump-leak system for photosynthate release is unlikely. A tentative model for photosynthate/proton symport not directly linked to proton pumping is presented as the mechanism of unloading.Abbreviations ABA abscisic acid - CCCP carbonyl cyanide m-chlorophenyl hydrazone - DTE diethioerythritol - FC fusicoccin - MES 2-(N-morpholino) ethanesulfonic acid monohydrate - NEM n-ethylmaleimide - PCMBS p-chloromercuriphenylsulfonic acid - TRIS 2-amino-2-(hydroxymethyl) propane-1,3 diol - VAN sodium orthovanadate     

Zn对细胞保护作用机理的研究

应用扫描质子微探针和同步辐射ｘ荧光分析技术测定了细胞中元素的分布和组成,为确定Zn是细胞结构成分提供了直接的实验依据.用上述核技术结合有关生化指标,分析测定了正常和损伤细胞（脂质过氧化损伤）中Fe,Zn和丙二醛、SH基含量变化的相互关系.实验结果表明,当细胞发生脂质过氧化损伤时,Fe含量和丙二醛含量同步增高,而Zn含量和SH基量则降低.给细胞补充Zn后,提高了细胞质膜中的Zn含量,SH基量也随之增加,同时丙二醛量降低.提示Zn保护细胞完整性的作用机理之一是控制脂质过氧化作用.Zn可保护膜蛋白的SH基,减少和阻止被Fe所催化的过氧化反应.     

The causes of altered chlorophyll fluorescence quenching induction in the Arabidopsis mutant lacking all minor antenna complexes

Non-photochemical quenching (NPQ) of chlorophyll fluorescence is the process by which excess light energy is harmlessly dissipated within the photosynthetic membrane. The fastest component of NPQ, known as energy-dependent quenching (qE), occurs within minutes, but the site and mechanism of qE remain of great debate. Here, the chlorophyll fluorescence of Arabidopsis thaliana wild type (WT) plants was compared to mutants lacking all minor antenna complexes (NoM). Upon illumination, NoM exhibits altered chlorophyll fluorescence quenching induction (i.e. from the dark-adapted state) characterised by three different stages: (i) a fast quenching component, (ii) transient fluorescence recovery and (iii) a second quenching component. The initial fast quenching component originates in light harvesting complex II (LHCII) trimers and is dependent upon PsbS and the formation of a proton gradient across the thylakoid membrane (ΔpH). Transient fluorescence recovery is likely to occur in both WT and NoM plants, but it cannot be overcome in NoM due to impaired ΔpH formation and a reduced zeaxanthin synthesis rate. Moreover, an enhanced fluorescence emission peak at ~679?nm in NoM plants indicates detachment of LHCII trimers from the bulk antenna system, which could also contribute to the transient fluorescence recovery. Finally, the second quenching component is triggered by both ΔpH and PsbS and enhanced by zeaxanthin synthesis. This study indicates that minor antenna complexes are not essential for qE, but reveals their importance in electron stransport, ΔpH formation and zeaxanthin synthesis.