Absence Of Mg2 Research Articles

Computation of energy interaction and intensity parameters for the complexation of Pr(III) with glutathione at different pH in the presence/absence of Mg2+: 4f-4f transition spectra as a probe

Free and complexed Pr3+ ion with reduced glutathione (GSH) have been studied in DMF and acetonitrile medium at different pH values (2, 4 and 6) in the presence and absence of Mg2+ ions through FTIR, comparative absorption spectroscopic analysis involving 4f-4f transition spectra. The interaction between the metal and ligand has been analyzed based on the computed values of different parameters, such as the Slater-Condon (Fk) parameter, Racah (Ek) factor, Lande (ξ4f) parameter, Nephelauxetic effect (β), bonding parameter (b1/2) percent covalency parameter (δ), oscillator strength (P) and Judd-Ofelt parameters (Tλ, λ = 2,4,6).

Examination of the Role of Mg2+ in the Mechanism of Nucleotide Binding to the Monomeric YME1L AAA+ Domain.

The first line of defense in the mitochondrial quality control network involves the stress response from a family of ATP-dependent proteases. We have reported that a solubilized version of the mitochondrial inner membrane ATP-dependent protease YME1L displays nucleotide binding kinetics that are sensitive to the reactive oxygen species hydrogen peroxide under a limiting ATP concentration. Our observations were consistent with an altered YME1L conformational ensemble leading to increased nucleotide binding site accessibility under oxidative stress conditions. To examine this hypothesis further, we report here the results of a comprehensive study of the thermodynamic and kinetic properties underlying the binding of nucleoside di- and triphosphate to the isolated YME1L AAA+ domain (YME1L-AAA+). A combination of fluorescence titrations, molecular dynamics, and stopped-flow fluorescence experiments have demonstrated similarity between nucleotide binding behaviors for YME1L under oxidative conditions and the isolated AAA+ domain. Our data demonstrate that YME1L-AAA+ binds ATP and ADP with affinities equal to ∼30 and 5 μM, respectively, in the absence of Mg2+. We note a negative heterotropic linkage effect between Mg2+ and ATP that arises as the MgCl2 concentration is increased such that the affinity of YME1L-AAA+ for ATP decreases to ∼60 μM in the presence of 10 mM MgCl2. Molecular dynamics methods allow for structural rationalization by revealing condition-dependent conformational populations for YME1L-AAA+. Taken together, these data suggest a preliminary model in which YME1L modulates its affinity for the nucleotide to stabilize against degradation or instability inherent to such stress conditions.

Molecular mechanisms underlying the extreme mechanical anisotropy of the flaviviral exoribonuclease-resistant RNAs (xrRNAs)

Mechanical anisotropy is an essential property for many biomolecules to assume their structures, functions and applications, however, the mechanisms for their direction-dependent mechanical responses remain elusive. Herein, by using a single-molecule nanopore sensing technique, we explore the mechanisms of directional mechanical stability of the xrRNA1 RNA from ZIKA virus (ZIKV), which forms a complex ring-like architecture. We reveal extreme mechanical anisotropy in ZIKV xrRNA1 which highly depends on Mg2+ and the key tertiary interactions. The absence of Mg2+ and disruption of the key tertiary interactions strongly affect the structural integrity and attenuate mechanical anisotropy. The significance of ring structures in RNA mechanical anisotropy is further supported by steered molecular dynamics simulations in combination with force distribution analysis. We anticipate the ring structures can be used as key elements to build RNA-based nanostructures with controllable mechanical anisotropy for biomaterial and biomedical applications.

Identification of a double-stranded RNA-degrading nuclease influencing both ingestion and injection RNA interference efficiency in the red flour beetle Tribolium castaneum

RNA interference (RNAi) efficiency dramatically varies among different insects and among administration methods. Numerous studies have revealed that a poor RNAi response is usually associated with a high double-stranded RNA (dsRNA)-degrading activity. Using the red flour beetle Tribolium castaneum, we conducted genome-wide identification of genes encoding dsRNA-degrading nucleases of the DNA/RNA non-specific endonuclease superfamily. To achieve a robust RNAi response in T. castaneum, four dsRNase genes were identified in the genome that seemed to be the potential factors reducing RNAi efficacy. Analysis of biochemical properties revealed that optimal conditions for the dsRNA-degrading activity were alkaline (pH 8.0) in the absence of Mg2+ at 37 °C. The dsRNA-degrading activity was predominantly present in the gut, and via heterologous expression and RNAi experimentation, gut-specific TcdsRNase1 was confirmed as the major nuclease performing dsRNA degradation. After a knockdown of the TcdsRNase1 nuclease activity, RNAi efficiency improved from 38.6% to 58.9% and from 20.9% to 53.9% for injection and ingestion of dsRNA, respectively. Our results contribute to a comprehensive understanding of the mechanisms influencing dsRNA stability and even RNAi efficiency in T. castaneum and point to a good method for improving RNAi efficiency through downregulation of the relevant nuclease activity.

Improved nearest-neighbor parameters for the stability of RNA/DNA hybrids under a physiological condition.

The stability of Watson–Crick paired RNA/DNA hybrids is important for designing optimal oligonucleotides for ASO (Antisense Oligonucleotide) and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)–Cas9 techniques. Previous nearest-neighbour (NN) parameters for predicting hybrid stability in a 1 M NaCl solution, however, may not be applicable for predicting stability at salt concentrations closer to physiological condition (e.g. ∼100 mM Na+ or K+ in the presence or absence of Mg2+). Herein, we report measured thermodynamic parameters of 38 RNA/DNA hybrids at 100 mM NaCl and derive new NN parameters to predict duplex stability. Predicted ΔG°37 and Tm values based on the established NN parameters agreed well with the measured values with 2.9% and 1.1°C deviations, respectively. The new results can also be used to make precise predictions for duplexes formed in 100 mM KCl or 100 mM NaCl in the presence of 1 mM Mg2+, which can mimic an intracellular and extracellular salt condition, respectively. Comparisons of the predicted thermodynamic parameters with published data using ASO and CRISPR–Cas9 may allow designing shorter oligonucleotides for these techniques that will diminish the probability of non-specific binding and also improve the efficiency of target gene regulation.

Halocatena pleomorpha gen. nov. sp. nov., an extremely halophilic archaeon of family Halobacteriaceae isolated from saltpan soil.

A novel archaeal strain designated as SPP-AMP-1T was isolated from saltpan soil, using the serial dilution method on a halophilic archaeal medium supplemented with ampicillin. Cells were both rod-shaped and pleomorphic in nature, non-motile, unable to produce acid from a variety of sugars or grow anaerobically with different substrates (l-arginine) and electron acceptors (DMSO, nitrate). Optimal growth was observed at 42 °C, 3.4-4.2 M NaCl and pH 7.2. Cells did not lyse in distilled water and grew in the absence of Mg2+ ions. Phylogenetic analysis based on the sequences of 16S rRNA gene, amino acid sequence of β'-subunit of RNA polymerase and 400 conserved proteins retrieved from the whole genome assemblies showed that strain SPP-AMP-1T was distantly related to any existing genera within the family Halobacteriaceae. MK-8 was the only quinone detected. Polar lipid analysis showed a unique combination of diethyl derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, glycosyl-mannosyl-glucosyl diether and sulphated glycosyl-mannosyl-glucosyl diether as the major lipids. The G+C content of genomic DNA is 57.7 mol%. The phenotypic, phylogenetic and genomic data supported the concept of the novel genus status of strain SPP-AMP-1T in the family Halobacteriaceae for which the name Halocatena pleomorpha gen. nov., sp. nov., is proposed; the type strain is SPP-AMP-1T (=JCM 31368T=KCTC 4276T=MTCC 12579T).

Hydrated Silicate Layer Formation on Mica-Type Crystals.

This study aims to investigate the growth of a cation-exchangeable hydrated layer on the surface of mica-type silicates based on a synthetic fluorophlogopite and a natural muscovite. Through the reaction of a synthetic fluorophlogopite using LiF, MgCl2, and a silica sol in water at 373 K for 48 h in the presence of urea, a hydrated phyllosilicate was formed on the fluoromica. As a result of examining the reaction in the alkali solution in the absence of Mg2+, the uptake of the silica sol would be included as a chemical process to begin the crystallization on fluorophlogopite because the lithium and ammonium ions (generated by urea hydrolysis) are known to contribute to enhanced adsorption. We found that the urea hydrolysis increased the pH, which, in turn, assisted the formation of magnesium hydroxide after the isomorphic substitution of Li+ for Mg2+. Bridging tetrahedral SiO4 with a magnesium-lithium double hydroxide afforded a 1 nm silicate layer. This facilitated the hectorite-like hydrated silicate layer to adhere closely to both the crystal edge and the cleaved face of the synthetic mica, which was found to coat the surface homogeneously. Only surface crystals were found to form through this process. The layered silicates included exchangeable hydrated cations for the cation-exchange reactions to expand the interlayer space by a cationic surfactant, dimethyldistearylammonium. The layered silicate also adsorbed methylene blue as a cationic dye in the aqueous phase. Apart from fluoromica, the natural muscovite also provided the surface to grow hydrated silicate layers, as a crystal turned dense blue when reacted with methylene blue.

Diazoxide affects mitochondrial bioenergetics by the opening of mKATP channel on submicromolar scale

BackgroundCytoprotection afforded by mitochondrial ATP-sensitive K+-channel (mKATP-channel) opener diazoxide (DZ) largely depends on the activation of potassium cycle with eventual modulation of mitochondrial functions and ROS production. However, generally these effects were studied in the presence of Mg∙ATP known to block K+ transport. Thus, the purpose of our work was the estimation of DZ effects on K+ transport, K+ cycle and ROS production in rat liver mitochondria in the absence of Mg∙ATP.ResultsWithout Mg·ATP, full activation of native mKATP-channel, accompanied by the increase in ATP-insensitive K+ uptake, activation of K+-cycle and respiratory uncoupling, was reached at ≤0.5 μM of DZ,. Higher diazoxide concentrations augmented ATP-insensitive K+ uptake, but not mKATP-channel activity. mKATP-channel was blocked by Mg·ATP, reactivated by DZ, and repeatedly blocked by mKATP-channel blockers glibenclamide and 5-hydroxydecanoate, whereas ATP-insensitive potassium transport was blocked by Mg2+ and was not restored by DZ. High sensitivity of potassium transport to DZ in native mitochondria resulted in suppression of mitochondrial ROS production caused by the activation of K+-cycle on sub-micromolar scale. Based on the oxygen consumption study, the share of mKATP-channel in respiratory uncoupling by DZ was found.ConclusionsThe study of mKATP-channel activation by diazoxide in the absence of MgATP discloses novel, not described earlier, aspects of mKATP-channel interaction with this drug. High sensitivity of mKATP-channel to DZ results in the modulation of mitochondrial functions and ROS production by DZ on sub-micromolar concentration scale. Our experiments led us to the hypothesis that under the conditions marked by ATP deficiency affinity of mKATP-channel to DZ can increase, which might contribute to the high effectiveness of this drug in cardio- and neuroprotection.

Small-angle Neutron Scattering Shows that the Solution Structures of the Bacterial Mg2+-Channel CorA are Overall Similar with and without Mg2+Bound

CorA is a homopentameric ion-channel involved in Mg2+-regulation in prokaryotes. In several crystal structures, CorA adapts a highly symmetric pentamer structure (closed form) in presence of Mg2+. Based on recent cryo electron microscopy work, it has been suggested that symmetry break is the main mechanism for regulation of the transport Mg2+ and for forming the open form of CorA. Here we used Small-Angle Neutron Scattering (SANS) and very recently developed neutron-invisible DDM micelles and nanodiscs to interrogate the solution structure of CorA in presence and absence of Mg2+.

Patch-Clamp Fluorometry Defines a Role for SUR1 in Nucleotide Inhibition of KATP Channels

ATP sensitive K+ (KATP) channels couple a cell's metabolic state to its electrical excitability. KATP is a hetero-octameric complex, with four pore-forming inward rectifier K+ channel subunits (Kir6.1 or Kir6.2), each associated with a modulatory sulfonylurea receptor (SUR1 or SUR2A/B). ATP/ADP binding to a site on the Kir6.2 subunit shuts KATP. In the presence of Mg2+, nucleotide binding to SUR1 stimulates KATP. However, SUR1 also has direct effects on gating at Kir6.2 in the absence of Mg2+. The presence of SUR1 increases channel open probability, but paradoxically also increases the apparent affinity for nucleotide inhibition at Kir6.2 by an unknown mechanism. We introduced the fluorescent non-canonical amino acid ANAP at a position (W311ANAP) in the cytoplasmic C-terminal region of Kir6.2 co-expressed with SUR1 in HEK 293T cells. This allowed us to simultaneously measure channel currents and inhibitory nucleotide binding to Kir6.2 via FRET between ANAP and a fluorescent ATP derivative (TNP-ATP). We fit models to the combined data sets to produce well-constrained estimates of open-state nucleotide affinity and the strength of coupling between nucleotide binding and channel closure. Our models suggest that “wild-type” KATP is closed when only one of its nucleotide binding sites is occupied. A Kir6.2 mutation (C166S) that profoundly increases channel activity had very little effect on nucleotide binding, but greatly perturbed the ability of bound nucleotide to inhibit KATP. In contrast, mutations at position E205 on SUR1, which is located on an intracellular loop close to the inhibitory site on Kir6.2, affected both TNP-ATP affinity and the ability of bound nucleotide to inhibit KATP. This suggests a dual role for SUR1 in KATP inhibition, both in contributing to nucleotide binding to the open state and in stabilizing the nucleotide-bound closed state.

Nucleotide inhibition of the pancreatic ATP-sensitive K+ channel explored with patch-clamp fluorometry.

Pancreatic ATP-sensitive K+ channels (KATP) comprise four inward rectifier subunits (Kir6.2), each associated with a sulphonylurea receptor (SUR1). ATP/ADP binding to Kir6.2 shuts KATP. Mg-nucleotide binding to SUR1 stimulates KATP. In the absence of Mg2+, SUR1 increases the apparent affinity for nucleotide inhibition at Kir6.2 by an unknown mechanism. We simultaneously measured channel currents and nucleotide binding to Kir6.2. Fits to combined data sets suggest that KATP closes with only one nucleotide molecule bound. A Kir6.2 mutation (C166S) that increases channel activity did not affect nucleotide binding, but greatly perturbed the ability of bound nucleotide to inhibit KATP. Mutations at position K205 in SUR1 affected both nucleotide affinity and the ability of bound nucleotide to inhibit KATP. This suggests a dual role for SUR1 in KATP inhibition, both in directly contributing to nucleotide binding and in stabilising the nucleotide-bound closed state.

ATP-dependent thermostabilization of human P-glycoprotein (ABCB1) is blocked by modulators.

P-glycoprotein (P-gp), an ATP-binding cassette transporter associated with multidrug resistance in cancer cells, is capable of effluxing a number of xenobiotics as well as anticancer drugs. The transport of molecules through the transmembrane (TM) region of P-gp involves orchestrated conformational changes between inward-open and inward-closed forms, the details of which are still being worked out. Here, we assessed how the binding of transport substrates or modulators in the TM region and the binding of ATP to the nucleotide-binding domains (NBDs) affect the thermostability of P-gp in a membrane environment. P-gp stability after exposure at high temperatures (37-80°C) was assessed by measuring ATPase activity and loss of monomeric P-gp. Our results show that P-gp is significantly thermostabilized (>22°C higher IT50) by the binding of ATP under non-hydrolyzing conditions (in the absence of Mg2+). By using an ATP-binding-deficient mutant (Y401A) and a hydrolysis-deficient mutant (E556Q/E1201Q), we show that thermostabilization of P-gp requires binding of ATP to both NBDs and their dimerization. Additionally, we found that transport substrates do not affect the thermal stability of P-gp either in the absence or presence of ATP; in contrast, inhibitors of P-gp including tariquidar and zosuquidar prevent ATP-dependent thermostabilization in a concentration-dependent manner, by stabilizing the inward-open conformation. Altogether, our data suggest that modulators, which bind in the TM regions, inhibit ATP hydrolysis and drug transport by preventing the ATP-dependent dimerization of the NBDs of P-gp.

NMR Chemical Exchange Measurements Reveal That N6-Methyladenosine Slows RNA Annealing.

N6-Methyladenosine (m6A) is an abundant epitranscriptomic modification that plays important roles in many aspects of RNA metabolism. While m6A is thought to mainly function by recruiting reader proteins to specific RNA sites, the modification can also reshape RNA-protein and RNA-RNA interactions by altering RNA structure mainly by destabilizing base pairing. Little is known about how m6A and other epitranscriptomic modifications might affect the kinetic rates of RNA folding and other conformational transitions that are also important for cellular activity. Here, we used NMR R1ρ relaxation dispersion and chemical exchange saturation transfer to noninvasively and site-specifically measure nucleic acid hybridization kinetics. The methodology was validated on two DNA duplexes and then applied to examine how a single m6A alters the hybridization kinetics in two RNA duplexes. The results show that m6A minimally impacts the rate constant for duplex dissociation, changing koff by ∼1-fold but significantly slows the rate of duplex annealing, decreasing kon by ∼7-fold. A reduction in the annealing rate was observed robustly for two different sequence contexts at different temperatures, both in the presence and absence of Mg2+. We propose that rotation of the N6-methyl group from the preferred syn conformation in the unpaired nucleotide to the energetically disfavored anti conformation required for Watson-Crick pairing is responsible for the reduced annealing rate. The results help explain why in mRNA m6A slows down tRNA selection and more generally suggest that m6A may exert cellular functions by reshaping the kinetics of RNA conformational transitions.

Regioselective formation of RNA strands in the absence of magnesium ions.

The oligomerization of ribonucleotides can produce short RNA strands in the absence of enzymes. This reaction gives one of two regioisomeric phosphodiester linkages, a 2′,5′- or a 3′,5′-diester. The former, non-natural linkage is detrimental for duplex stability, and is known to form preferentially in oligomerizations occurring in homogeneous solution with preactivated nucleotides in the presence of magnesium cations. We have studied ribonucleotide oligomerization with in situ activation, using NMR as monitoring technique. Unexpectedly, the known preference for 2′,5′-linkages in the oligomerization of AMP was reversed in the absence of magnesium ions at slightly basic pH. Further, oligomerization was surprisingly efficient in the absence of Mg2+ salts, producing oligomers long enough for duplex formation. A quantitative systems chemistry analysis then revealed that the absence of magnesium ions favors the activation of nucleotides, and that the high concentration of active species can compensate for slower coupling. Further, organocatalytic intermediates can help to overcome the unfavorable regioselectivity of the magnesium-catalyzed reactions. Our findings allay concerns that RNA may have been difficult to form in the absence of enzymes. They also show that there is an efficient path to genetic material that does not require mineral surfaces or cations known to catalyze RNA hydrolysis.

Coral acid rich protein selects vaterite polymorph in vitro

Corals and other biomineralizing organisms use proteins and other molecules to form different crystalline polymorphs and biomineral structures. In corals, it’s been suggested that proteins such as Coral Acid Rich Proteins (CARPs) play a major role in the polymorph selection of their calcium carbonate (CaCO3) aragonite exoskeleton. To date, four CARPs (1–4) have been characterized: each with a different amino acid composition and different temporal and spatial expression patterns during coral developmental stages. Interestingly, CARP3 is able to alter crystallization pathways in vitro, yet its function in this process remains enigmatic. To better understand the CARP3 function, we performed two independent in vitro CaCO3 polymorph selection experiments using purified recombinant CARP3 at different concentrations and at low or zero Mg2+ concentration. Our results show that, in the absence of Mg2+, CARP3 selects for the vaterite polymorph and inhibits calcite. However, in the presence of a low concentration of Mg2+ and CARP3 both Mg-calcite and vaterite are formed, with the relative amount of Mg-calcite increasing with CARP3 concentration. In all conditions, CARP3 did not select for the aragonite polymorph, which is the polymorph associated to CARP3 in vivo, even in the presence of Mg2+ (Mg:Ca molar ratio equal to 1). These results further emphasize the importance of Mg:Ca molar ratios similar to that in seawater (Mg:Ca equal to 5) and the activity of the biological system in a aragonite polymorph selection in coral skeleton formation.

Against Expectations: Unassisted RNA Adsorption onto Negatively Charged Lipid Bilayers.

The composition and physicochemical properties of biological membranes can be altered by diverse membrane integral and peripheral proteins as well as by small molecules, natural and synthetic. Diverse oligonucleotides have been shown to electrostatically interact with cationic and bivalent ion loaded zwitterionic liposomes, leading to the formation of oligonucleotide-liposome aggregates. However, interaction of RNAs with other membrane surfaces remains ill understood. We used the nonnatural RNA10 to investigate RNA binding to anionic and net-uncharged membrane surfaces. RNA10 had initially been selected in a screen for nonnatural RNA motives that bind to phosphatidylcholine liposomes in the presence of Mg2+. Here we show that interaction of defined RNA molecules with membrane surfaces crucially depends on electrostatic surface properties. Furthermore, RNA10 electrostatically binds to anionic lipid bilayers in the absence of Mg2+ or other bivalent cations, and this interaction leads to measurably changed physicochemical properties of the bilayer and the oligonucleotide. Thus, the structure of polyanionic RNA can be modulated via contact with negatively charged membrane surfaces and vice versa.

Influence of Gelatin–Agarose Composites and Mg on Hydrogel-Carbonate Aggregate Formation and Architecture

Hydrogels are adequate systems for investigating biological structural material formation as they mimic a wide range of biomineralization conditions. Previous studies focused on the influence of single-component gel systems in the growth of calcite aggregates and their microstructure characteristics. In this work, we investigate the effects of hydrogel mixtures (gelatin/agarose), in the presence and absence of Mg in the growth medium, on calcite aggregate formation, hydrogel incorporation in the calcite, mode of crystallite assembly, and hierarchical mineral organization. We find marked differences between aggregates developed in gel mixtures with increased content of either gelatin or agarose. The presence of Mg, in addition to the gel incorporation, strongly influences the local lattice deformation within the aggregates. The mode of local deformation (homogeneous or localized) is closely related to the size, distribution, and crystallographic co-orientation of subunits within the aggregate. An increase in agarose induces homogeneous distribution of local deformation within the aggregates and formation of a graded mineral arrangement (archetypical spherulites), while an increase in gelatin leads to differentiation of the center and rim portions within the same aggregate. On a higher hierarchical level, when all subunits of an aggregate are considered, both agarose and gelatin evoke the formation of polycrystals.

ADSORPTION BEHAVIOR OF PROTEINS ON Mg(II)-DOPED CALCIUM HYDROXYAPATITE PARTICLES AT VARIOUS Mg(II) CONTENTS

In this study, fundamental experiments on the adsorption behavior of proteins on synthesized magnesium-doped calcium hydroxyapatite particles (MgHap) at various Mg/(Ca + Mg) molar ratios (abbreviated as XMg : 0–0.3) were examined. Rod-like calcium hydroxyapatite particles, with a width of 24 nm and length of 180 nm, were obtained in the absence of Mg2+ (XMg = 0). With the increase in XMg up to 0.10, the width and length of MgHap particles decreased to ~10 and 50 nm, respectively. Agglomerates of small thin particles were obtained at XMg ≥ 0.15. All of the synthesized particles exhibited single phase of calcium hydroxyapatite particles (CaHap). The XMg values for the particles precipitated at XMg ≥ 0.04 were less than those of the corresponding solutions. Hence, almost all of the MgHap particles are deficient in magnesium. We propose that to compensate for the cation deficiency, PO43- is incorporated as HPO42- and H2PO4- and thus decreases the particle negative charge. In addition, all of the adsorption isotherms of typical acidic and basic proteins such as bovine serum albumin (BSA) and lysozyme (LSZ) were compared. The isotherms obtained from a 1 × 10−4 mol/dm3 KCl solution were of the Langmuirian type. The saturated amount of adsorbed BSA (nsBSA) strongly depended on the particle length rather than on the XMg value. At a particle length of greater than 150 nm, nsBSA values abruptly increased. This result strongly suggests that the effects of the C sites produced on the ac and bc faces are advantageous for BSA adsorption. In other words, the adsorption of BSA on MgHap particles was considerably dependent on the length at a critical value of greater than 150 nm. However, the saturated amount of adsorbed LSZ (nsLSZ) exhibited no remarkable variation with the particle length.

Estimate of the Upper Limit on Hot Plasma Differential Emission Measure (DEM) in Non-Flaring Active Regions and Nanoflare Frequency Based on the Mg xii Spectroheliograph Data from CORONAS-F/SPIRIT

The nanoflare-heating theory predicts steady hot plasma emission in the non-flaring active regions. It is hard to find this emission with conventional non-monochromatic imagers (such as Atmospheric Imaging Assembly or X-Ray Telescope), because their images contain a cool temperature background. In this work, we search for hot plasma in non-flaring active regions using the Mg XII spectroheliograph onboard Complex Orbital Observations Near-Earth of Activity on the Sun (CORONAS)-F/SPectroheliographIc X-ray Imaging Telescope (SPIRIT). This instrument acquired monochromatic images of the solar corona in the Mg XII 8.42 \AA line, which emits only at temperatures higher than 4 MK. The Mg XII images contain the signal only from hot plasma without any low-temperature background. We studied the hot plasma in active regions using the SPIRIT data from 18-28 February 2002. During this period, the Mg XII spectroheliograph worked with a 105-second cadence almost without data gaps. The hot plasma was observed only in the flaring active regions. We do not observe any hot plasma in non-flaring active regions. The hot plasma column emission measure in the non-flaring active region should not exceed $3 \times 10^{24}$ cm$^{-5}$. The hot Differential Emission Measure (DEM) is less than 0.01 % of the DEM of the main temperature component. Absence of Mg XII emission in the non-flaring active regions can be explained by weak and frequent nanoflares (delay less than 500 seconds) or by very short and intense nanoflares that lead to non-equilibrium ionization.

Loss of cytosolic Mg2+ binding sites in the Thermotoga maritima CorA Mg2+ channel is not sufficient for channel opening

The CorA Mg2+ channel is a homopentamer with five-fold symmetry. Each monomer consists of a large cytoplasmic domain and two transmembrane helices connected via a short periplasmic loop. In the Thermotoga maritima CorA crystal structure, a Mg2+ is bound between D89 of one monomer and D253 of the adjacent monomer (M1 binding site). Release of Mg2+ from these sites has been hypothesized to cause opening of the channel. We generated mutants to disrupt Mg2+ interaction with the M1 site. Crystal structures of the D89K/D253K and D89R/D253R mutants, determined to 3.05 and 3.3 Å, respectively, showed no significant structural differences with the wild type structure despite absence of Mg2+ at the M1 sites. Both mutants still appear to be in the closed state. All three mutant CorA proteins exhibited transport of 63Ni2+, indicating functionality. Thus, absence of Mg2+ from the M1 sites neither causes channel opening nor prevents function. We also provide evidence that the T. maritima CorA is a Mg2+ channel and not a Co2+ channel.