Charge selectivity of the Cys-loop family of ligand-gated ion channels

The determinants of charge selectivity of the Cys-loop family of ligand-gated ion channels have been studied for more than a decade. The investigations have mainly covered homomeric receptors e.g. the nicotinic acetylcholine receptor alpha7, the glycine receptor alpha1 and the serotonin receptor 5-HT(3A). Only recently, the determinants of charge selectivity of heteromeric receptors have been addressed for the GABA(A) receptor alpha2beta3gamma2. For all receptor subtypes, the selectivity determinants have been located to an intracellular linker between transmembrane domains M1 and M2. Two features of the M1-M2 linker appear to control ion selectivity. A central role for charged amino acid residues in selectivity has been almost universally observed. Furthermore, recent studies point to an important role of the size of the narrowest constriction in the pore. In the present review, these determinants of charge selectivity of the Cys-loop family of ligand-gated ion channels will be discussed in detail.     

Variation of charge transfer kinetics in structurally closely related dyads with rhenium photosensitizers

The kinetics for photoinduced charge separation and thermal charge-recombination processes in three donor-bridge-acceptor molecules with rhenium(I) tricarbonyl diimine photosensitizers were investigated. Time resolved luminescence and transient absorption spectroscopies reveal that in addition to driving force effects, differences in bridge-mediated electronic donor-acceptor coupling among the three dyads play important roles. Notably, it is seen that charge separation depends strongly on whether initial photoexcitation involves promotion of an electron towards or away from a phenothiazine electron donor. Thermal charge recombination rates are found to differ by a factor of 2 between two isomeric dyads due to an electronic coupling effect.     

Space charge drift from a +/- 400-kV direct current transmission line

The Minnesota Environmental Quality Board completed a 3-year study of the space charge plume outside the right of way of a bipolar (+/- 400 kV) direct current transmission line in rural west central Minnesota. The purpose of the study was to determine the levels of DC potential gradient and small airs ions in the downwind plume to which humans and animals may be exposed. Potential gradient measurements show that a space charge plume is detectable downwind at least 1600 m from the transmission line. Plume relaxation rates indicate that under certain conditions one could detect the plume at twice that distance or more. Net charge in the plume assumes the polarity of the downwind energized transmission line conductor. The bipolar DC line thus electrically bisects the land through which it passes; on one side of the line there is a net positive space charge, on the other side a net negative space charge. Electric charge in the plume resides on aerosols and small air ions. Polar conductivity data substantiate the fact that small air ions of one polarity in the plume are elevated while those of opposite polarity are suppressed compared to background concentrations found in the rural environment. The resulting charge imbalance persists downwind at least 1600 m, though the median small air ion concentrations of plume polarity have adjusted to within the background range by that time with perpendicular wind speed components between 1 and 6 m/sec.     

Intracellular sorting of the tail-anchored protein cytochrome b5 in plants: a comparative study using different isoforms from rabbit and Arabidopsis

Tail-anchored (TA) proteins are bound to membranes by a hydrophobic sequence located very close to the C-terminus, followed by a short luminal polar region. Their active domains are exposed to the cytosol. TA proteins are synthesized on free cytosolic ribosomes and are found on the surface of every subcellular compartment, where they play various roles. The basic mechanisms of sorting and targeting of TA proteins to the correct membrane are poorly characterized. In mammalian cells, the net charge of the luminal region determines the sorting to the correct target membrane, a positive charge leading to mitochondria and negative or null charge to the endoplasmic reticulum (ER). Here sorting signals of TA proteins were studied in plant cells and compared with those of mammalian proteins, using in vitro translation-translocation and in vivo expression in tobacco protoplasts or leaves. It is shown that rabbit cytochrome b5 (cyt b5) with a negative charge is faithfully sorted to the plant ER, whereas a change to a positive charge leads to chloroplast targeting (instead of to mitochondria as observed in mammalian cells). The subcellular location of two cyt b5 isoforms from Arabidopsis thaliana (At1g26340 and At5g48810, both with positive net charge) was then determined. At5g48810 is targeted to the ER, and At1g26340 to the chloroplast envelope. The results show that the plant ER, unlike the mammalian ER, can accommodate cytochromes with opposite C-terminal net charge, and plant cells have a specific and as yet uncharacterized mechanism to sort TA proteins with the same positive C-terminal charge to different membranes.     

Separation of intramembrane charging components in low-calcium solutions in frog skeletal muscle

The inactivation of charge movement components by small (-100 to -70 mV) shifts in holding potential was examined in voltage-clamped intact amphibian muscle fibers in low [Ca2+], Mg(2+)-containing solutions. The pulse protocols used both large voltage excursions and smaller potential steps that elicited prolonged (q gamma) transients. Charge species were distinguished through the pharmacological effects of tetracaine. These procedures confirmed earlier observations in cut fibers and identified the following new properties of the q gamma charge. First, q gamma, previously defined as the tetracaine-sensitive charge, is also the component primarily responsible for the voltage-dependent inactivation induced by conditions of low extracellular [Ca2+]. Second, this inactivation separates a transient that includes a "hump" component and which has kinetics and a voltage dependence distinct from the monotonic decay that remains. Third, q gamma, previously associated with delayed charge movements, can also contribute significant charge transfer at early times. These findings suggest that the parallel inhibition of calcium signals and charge movements reported in low [Ca2+] solutions arises from influences on q gamma charge (Brum et al., 1988a, b). They also reconcile reports that implicate tetracaine-sensitive (q gamma) charge in excitation-contraction coupling with evidence that early intramembrane events are also involved in this process (Pizarro et al., 1989). Finally, they are relevant to hypotheses of possible feedback or feed-forward roles of q gamma in excitation-contraction coupling.     

Genome-wide screening of yeast metal homeostasis genes involved in mitochondrial functions

Metal ions are essential for mitochondria to execute their roles. Yeast mutants that are sensitive to metals (either excess or deficiency) on non-fermentable media but not on fermentable media may carry mutations in genes that participate in metal homeostasis involving mitochondrial functions. A collection of approximately 4,800 haploid yeast deletion mutants was screened for metal ion homeostasis genes linked to mitochondrial respiration. In addition to several well-characterized metal homeostasis genes, 45 new mutants, impaired in various molecular functions, were identified on non-fermentable media that were sensitive to adscititious metals or metal deficiency. While 35 of these mutants displayed metal-sensitivity only on non-fermentable media, the remaining 10 also exhibited metal sensitivity on fermentable media, suggesting metal-sensitivity of the latter is not due to mitochondrial dysfunction. Inductively coupled plasma optical emission spectrometry (ICP-OES) was conducted for 12 mutants that were sensitive to metal excess to analyze their metal contents. Among these 12 mutants 7 were sensitive to metal excess on non-fermentable but not on fermentable media. All the seven respiration-dependent mutants displayed abnormal levels of metal ions inside mitochondria, indicative of disrupted mitochondrial metal homeostasis. This study therefore effectively identified multiple new genes involved in metal homeostasis pathways possibly pertinent to mitochondrial functions, and should be helpful for future studies to further understand their molecular roles.     

An All‐Organic Non‐aqueous Lithium‐Ion Redox Flow Battery

A non‐aqueous lithium‐ion redox flow battery employing organic molecules is proposed and investigated. 2,5‐Di‐tert‐butyl‐1,4‐bis(2‐methoxyethoxy)benzene and a variety of molecules derived from quinoxaline are employed as initial high‐potential and low‐potential active materials, respectively. Electrochemical measurements highlight that the choice of electrolyte and of substituent groups can have a significant impact on redox species performance. The charge‐discharge characteristics are investigated in a modified coin‐cell configuration. After an initial break‐in period, coulombic and energy efficiencies for this unoptimized system are ～70% and ～37%, respectively, with major charge and discharge plateaus between 1.8‐2.4 V and 1.7‐1.3 V, respectively, for 30 cycles. Performance enhancements are expected with improvements in cell design and materials processing.     

Charged extracellular residues, conserved throughout a G-protein-coupled receptor family, are required for ligand binding, receptor activation, and cell-surface expression

For G-protein-coupled receptors (GPCRs) in general, the roles of extracellular residues are not well defined compared with residues in transmembrane helices (TMs). Nevertheless, extracellular residues are important for various functions in both peptide-GPCRs and amine-GPCRs. In this study, the V(1a) vasopressin receptor was used to systematically investigate the role of extracellular charged residues that are highly conserved throughout a subfamily of peptide-GPCRs, using a combination of mutagenesis and molecular modeling. Of the 13 conserved charged residues identified in the extracellular loops (ECLs), Arg(116) (ECL1), Arg(125) (top of TMIII), and Asp(204) (ECL2) are important for agonist binding and/or receptor activation. Molecular modeling revealed that Arg(125) (and Lys(125)) stabilizes TMIII by interacting with lipid head groups. Charge reversal (Asp(125)) caused re-ordering of the lipids, altered helical packing, and increased solvent penetration of the TM bundle. Interestingly, a negative charge is excluded at this locus in peptide-GPCRs, whereas a positive charge is excluded in amine-GPCRs. This contrasting conserved charge may reflect differences in GPCR binding modes between peptides and amines, with amines needing to access a binding site crevice within the receptor TM bundle, whereas the binding site of peptide-GPCRs includes more extracellular domains. A conserved negative charge at residue 204 (ECL2), juxtaposed to the highly conserved disulfide bond, was essential for agonist binding and signaling. Asp(204) (and Glu(204)) establishes TMIII contacts required for maintaining the beta-hairpin fold of ECL2, which if broken (Ala(204) or Arg(204)) resulted in ECL2 unfolding and receptor dysfunction. This study provides mechanistic insight into the roles of conserved extracellular residues.     

Expression and characterization of human bifunctional peptidylglycine alpha-amidating monooxygenase

We report the purification and characterization of human bifunctional peptidylglycine alpha-amidating monooxygenase (the bifunctional PAM) expressed in Chinese hamster ovary cells. PAM is in charge of the formation of the C-terminal amides of biologically active peptides. The bifunctional PAM possesses two catalytic domains in a single polypeptide, peptidylglycine alpha-hydroxylating monooxygenase (PHM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PAL, EC 4.3.2.5). By introducing a stop codon at 835 Glu, we were able to eliminate the membrane-spanning domain in the C-terminal region and succeeded in purifying a soluble form of bifunctional PAM that was secreted into the medium. Through a three-step purification procedure, we obtained 0.3mg of the purified PAM, which showed a single band at 91 kDa on SDS-PAGE, from 1L of monolayer culture medium. Metals contained in the purified PAM were analyzed and chemical modifications were performed to gain insight into the mechanism of the PAL reaction. Inductively coupled plasma detected 0.62 mol of Zn(2+) and 1.25 mol of Cu(2+) per mol of bifunctional PAM. Further, the addition of 1mM EDTA reduced the PAL activity by about 50%, but the decreased activity was recovered by the addition of an excess amount of Zn(2+). In a series of chemical modifications, phenylglyoxal almost completely eliminated the PAL activity and diethyl pyrocarbonate suppressed activity by more than 70%. These findings implied that Arg and His residues might play crucial roles during catalysis.     

Effects of aminooxy analogues of biogenic polyamines on aggregation and stability of calf thymus DNA

The effect of a series of aminooxy analogues of the biogenic polyamines spermidine and spermine on the conformation of calf thymus DNA is studied. These new molecules are isosteric and charge insufficient analogues that are suitable to study the roles of both charge distribution and structural requirements in the molecular physiology of the biogenic polyamines. They are also evidenced as useful tools to inhibit polyamine biosynthesis and cell growth. Circular dichroism (CD) spectra of solutions containing DNA and the aminooxy analogues at different concentrations (100-1000 microM) and different pH values, (5-7.5) are recorded. We use both sonicated and highly polymerized calf thymus DNA. The CD spectra of sonicated DNA showed the formation of Psi-DNA, a highly ordered aggregated structure similar to liquid crystals, in the presence of the aminooxy analogues. Aggregation induced by an aminooxy derivative of spermine is followed by DNA collapse when increasing the polyamine concentration. The features of Psi-DNA are not detected for highly polymerized DNA. Temperature melting measurements support a high degree of structural order of the aggregates. The CD experiments indicate that dications are unable to induce major changes on the macromolecular structure of DNA. In addition, aggregation is only observed when the trimethylene moiety is present between two adjacent positive charges. The observed differences among the CD spectra of DNA solutions with different aminooxy derivatives of spermidine indicate different roles for different amino groups of this biogenic polyamine when interacting with DNA. Our results support the idea that aminooxy analogues can be used as good models in studying the physiological functions of biogenic polyamines.     

Effects of direct current on dog liver: Possible mechanisms for tumor electrochemical treatment

Mechanisms of tumor electrochemical treatment (ECT) were studied using normal dog liver. Five physical and chemical methods were used. Two platinum electrodes were inserted into an anesthetized dog's liver at 3 cm separation. A voltage of 8.5 V direct current (DC) at an average current of 30 mA was applied for 69 min; total charge was 124 coulombs. Concentrations of selected ions near the anode and cathode were measured. The concentrations of Na⁺ and K⁺ ions were higher around the cathode, whereas the concentration of Cl⁻ ions was higher around the anode. Water contents and pH were determined near the anode and the cathode at the midpoint between the two electrodes and in an untreated area away from the electrodes. Hydration occurred around the cathode, and dehydration occurred around the anode. The pH values were 2.1 near the anode and 12.9 near the cathode. Spectrophotometric scans of the liver sample extract were obtained, and the released gases were identified by gas chromatography as chlorine at the anode and hydrogen at the cathode. These results indicate that a series of electrochemical reactions take place during ECT. The cell metabolism and its environment are severely disturbed. Both normal and tumor cells are rapidly and completely destroyed in this altered environment. We believe that the above reactions are the ECT mechanisms for treating tumors. Bioelectromagnetics 18:2–7, 1997. © 1997 Wiley-Liss, Inc.     

Formation of supported phospholipid bilayers on molecular surfaces: role of surface charge density and electrostatic interaction

Electrostatic interaction is known to play important roles in the adsorption of charged lipids on oppositely charged surfaces. Here we show that, even for charge neutral (zwitterionic) lipids, electrostatic interaction is critical in controlling the adsorption and fusion of lipid vesicles to form supported phospholipid bilayers (SPBs) on surfaces. We use terminally functionalized alkanethiol self-assembled monolayers (SAMs) to systematically control the surface charge density. Charge neutral egg phophatidylcholine (eggPC) vesicles readily fuse into SPBs on either a positively charged 11-aminino-1-undecanethiol SAM or a negatively charged 10-carboxy-1-decanethiol SAM when the density of surface charge groups is > or = 80%. These processes depend critically on the buffer environment: fusion of adsorbed vesicles to form SPBs on each charged molecular surface does not occur when the molecular ion of the buffer used is of the opposite charge type. We attribute this to the high entropic repulsion (electric double layer repulsion) due to the large size of molecular counterions. On the other hand, such a critical dependence on buffer type is not observed when charged lipids are used. This study suggests the general importance of controlling electrostatic interaction in the formation of stable SPBs.     

The Roles of Magnesium in Biotechnology

Abstract

This review highlights the important roles played by magnesium in the growth and metabolic functions of microbial and animal cells, and therefore assigns a key role for magnesium ions in biotechnology. The fundamental biochemical and physiological actions of magnesium as a regulatory cation are outlined. Such actions are deemed to be relevant in an applied sense, because Mg²⁺ availability in cell culture and fermentation media can dramatically influence growth and metabolism of cells. Manipulation of extracellular and intracellular magnesium ions can thus be envisaged as a relatively simplistic, but nevertheless versatile, means of physiological cell engineering. In addition, biological antagonism between calcium and magnesium at the molecular level may have profound consequences for the optimization of biotechnological processes that exploit cells. In fermentation, for example, it is argued that the efficiency of microbial conversion of substrate to product may be improved by altering Mg:Ca concentration ratios in industrial feedstocks in a way that makes more magnesium available to the cells. With particular respect to yeast-based biotechnologies, magnesium availability is seen as being crucially important in governing central pathways of carbohydrate catabolism, especially ethanolic fermentation. It is proposed that such influences of magnesium ions are expressed at the combined levels of key enzyme activation and cell membrane stabilization. The former ensures optimum flow of substrate to ethanol and the latter acts to protect yeasts from physical and chemical stress.     

Speciation studies of ternary complexes of some essential divalent metal ions with L-histidine and L-glutamic acid in DMSO-water mixtures

Abstract

Chemical speciation of ternary complexes of Ca(II), Mg(II) and Zn(II) ions with L-histidine as the primary ligand (L) and L-glutamic acid as the secondary ligand (X) has been studied pH metrically in the concentration range of 0.0-60.0% v/v DMSO-water mixtures maintaining an ionic strength of 0.16 mol L^-1 using sodium chloride at 303.0 K. Titrations were carried out in different relative concentrations (M:L:X = 1.0:2.5:2.5, 1.0:2.5:5.0, 1.0:5.0:2.5) of metal (M) to L-histidine to L-glutamic acid with sodium hydroxide. Stability constants of ternary complexes were refined with MINIQUAD75. The best-fit chemical models were selected based on statistical parameters and residual analysis. The predominant species detected for Ca(II), Mg(II) and Zn(II) are ML₂XH₂, MLXH₂ and MLX₂. Extra stability of ternary complexes compared to their binary complexes was explained to be due to electrostatic interactions of the side chains of ligands, charge neutralisation, chelate effect, stacking interactions and hydrogen bonding. The species distribution with pH at different compositions of DMSO and the plausible equilibria for the formation of species are discussed.     

规模化蛋白质鉴定中母离子的准确检测技术研究

蛋白质组学的基础研究之一是蛋白质鉴定.规模化的蛋白质鉴定通常采用"鸟枪法",即选择一些酶切肽段(母离子)碎裂生成二级谱图,通过二级谱图及其母离子质量鉴定肽段,再推断对应的蛋白质.在鉴定过程中,母离子质量是一个关键参数.母离子是否是肽段的单同位素峰决定了正确肽段是否能进入候选,母离子的质量精度决定了候选肽段的数目.本文从判断单同位素峰和系统误差校准这两个角度研究了母离子的准确检测技术.判断单同位素峰的技术在蛋白质上已有研究,包括电荷判断、单同位素峰判断和重叠同位素峰判断.可以借鉴蛋白质水平的技术研究母离子的单同位素峰判断方法.同时母离子的系统误差校准也有较为成熟的方法.这两个角度的研究有助于提高规模化蛋白质的鉴定率.     

Brassinosteroids function in spikelet differentiation and degeneration in rice

Brassinosteroids(BRs) play crucial roles in many aspects of plant development. However, their function in spikelet differentiation and degeneration in rice(Oryza sativa L.) remains unclear. Here, we investigated the roles of these phytohormones in spikelet development in fieldgrown rice subjected to five different nitrogen(N)fertilization treatments during panicle differentiation. BR levels and expression of genes involved in BR biosynthesis and signal transduction were measured in spikelets. Pollen fertility and the number of differentiated spikelets were closely associated with 24-epicastasterone(24-epiCS) and28-homobrassinolide(28-homoBL) levels in spikelets.Enhanced BR biosynthesis and signal transduction, in response to N treatment, enhanced spikelet differentiation, reduced spikelet degeneration, and increased grain yield. Increases in proton-pumping ATPase activity, ATPconcentration, energy charge, and antioxidant system(AOS) levels were consistent with 24-epiCS and28-homoBL concentrations. Exogenous application of24-epiCS or 28-homoBL on young panicles induced a marked increase in endogenous 24-epiCS or 28-homoBL levels, energy charge, AOS levels, spikelet differentiation, and panicle weight. The opposite effects were observed following treatment with a BR biosynthesis inhibitor. Our findings indicate that, in rice, BRs mediate the effects of N fertilization on spikelet development and play a role in promoting spikelet development through increasing AOS levels and energy charge during panicle development.     

On the Molecular Origin of Charge Separation at the Donor–Acceptor Interface

Fullerene‐based acceptors have dominated organic solar cells for almost two decades. It is only within the last few years that alternative acceptors rival their dominance, introducing much more flexibility in the optoelectronic properties of these material blends. However, a fundamental physical understanding of the processes that drive charge separation at organic heterojunctions is still missing, but urgently needed to direct further material improvements. Here a combined experimental and theoretical approach is used to understand the intimate mechanisms by which molecular structure contributes to exciton dissociation, charge separation, and charge recombination at the donor–acceptor (D–A) interface. Model systems comprised of polythiophene‐based donor and rylene diimide‐based acceptor polymers are used and a detailed density functional theory (DFT) investigation is performed. The results point to the roles that geometric deformations and direct‐contact intermolecular polarization play in establishing a driving force (energy gradient) for the optoelectronic processes taking place at the interface. A substantial impact for this driving force is found to stem from polymer deformations at the interface, a finding that can clearly lead to new design approaches in the development of the next generation of conjugated polymers and small molecules.     

Regulation of Escherichia coli polynucleotide phosphorylase by ATP

Polynucleotide phosphorylase (PNPase), an enzyme conserved in bacteria and eukaryotic organelles, processively catalyzes the phosphorolysis of RNA, releasing nucleotide diphosphates, and the reverse polymerization reaction. In Escherichia coli, both reactions are implicated in RNA decay, as addition of either poly(A) or heteropolymeric tails targets RNA to degradation. PNPase may also be associated with the RNA degradosome, a heteromultimeric protein machine that can degrade highly structured RNA. Here, we report that ATP binds to PNPase and allosterically inhibits both its phosphorolytic and polymerization activities. Our data suggest that PNPase-dependent RNA tailing and degradation occur mainly at low ATP concentrations, whereas other enzymes may play a more significant role at high energy charge. These findings connect RNA turnover with the energy charge of the cell and highlight unforeseen metabolic roles of PNPase.     

Protein diffusion in crowded electrolyte solutions

We report on a combined cold neutron backscattering and spin-echo study of the short-range and long-range nanosecond diffusion of the model globular protein bovine serum albumin (BSA) in aqueous solution as a function of protein concentration and NaCl salt concentration. Complementary small angle X-ray scattering data are used to obtain information on the correlations of the proteins in solution. Particular emphasis is put on the effect of crowding, i.e. conditions under which the proteins cannot be considered as objects independent of each other. We thus address the question at which concentration this crowding starts to influence the static and in particular also the dynamical behaviour. We also briefly discuss qualitatively which charge effects, i.e. effects due to the interplay of charged molecules in an electrolyte solution, may be anticipated. Both the issue of crowding as well as that of charge effects are particularly relevant for proteins and their function under physiological conditions, where the protein volume fraction can be up to approximately 40% and salt ions are ubiquitous. The interpretation of the data is put in the context of existing studies on related systems and of existing theoretical models.