Absence Of Mg2 Research Articles

Dantrolene requires Mg2+ to arrest malignant hyperthermia

Malignant hyperthermia (MH) is a clinical syndrome of skeletal muscle that presents as a hypermetabolic response to volatile anesthetic gases, where susceptible persons may develop lethally high body temperatures. Genetic predisposition mainly arises from mutations on the skeletal muscle ryanodine receptor (RyR). Dantrolene is administered to alleviate MH symptoms, but its mechanism of action and its influence on the Ca2+ transients elicited by MH triggers are unknown. Here, we show that Ca2+ release in the absence of Mg2+ is unaffected by the presence of dantrolene but that dantrolene becomes increasingly effective as cytoplasmic-free [Mg2+] (free [Mg2+]cyto) passes mM levels. Furthermore, we found in human muscle susceptible to MH that dantrolene was ineffective at reducing halothane-induced repetitive Ca2+ waves in the presence of resting levels of free [Mg2+]cyto (1 mM). However, an increase of free [Mg2+]cyto to 1.5 mM could increase the period between Ca2+ waves. These results reconcile previous contradictory reports in muscle fibers and isolated RyRs, where Mg2+ is present or absent, respectively, and define the mechanism of action of dantrolene is to increase the Mg2+ affinity of the RyR (or "stabilize" the resting state of the channel) and suggest that the accumulation of the metabolite Mg2+ from MgATP hydrolysis is required to make dantrolene administration effective in arresting an MH episode.

Sulfate removal from wastewater using ettringite precipitation: Magnesium ion inhibition and process optimization

One of the main challenges in industrial wastewater treatment and recovery is the removal of sulfate, which usually coexists with Ca2+ and Mg2+. The effect of Mg2+ on sulfate removal by ettringite precipitation was investigated, and the process was optimized in the absence and presence of Mg2+. In the absence of Mg2+, the optimum conditions with sulfate removal of 99.7% were obtained at calcium-to-sulfate ratio of 3.20, aluminum-to-sulfate ratio of 1.25 and pH of 11.3 using response surface methodology. In the presence of Mg2+, sulfate removal efficiency decreased with increasing Mg2+ concentration, and the inhibitory effect of Mg2+ matched the competitive inhibition Monod model with half maximum inhibition concentration of 57.4 mmol/L. X-ray diffraction and Fourier transform infrared spectroscopy analyses of precipitates revealed that ettringite was converted to hydrotalcite-type (HT) compound in the presence of Mg2+. The morphology of precipitates was transformed from prismatic crystals to stacked layered crystals, which confirmed that Mg2+ competes with Ca2+ for Al3+ to form HT compound. A two-stage process was designed with Mg2+ removal before ettringite precipitation to eliminate the inhibitory effect, and is potential to realize sludge recovery at the same time of effective removal of sulfate and hardness.

Effect of Hardness Ions on Cr(VI) Reduction with Organic Reducing Substances

Batch laboratory-scale experiments were conducted to investigate the effects of hardness ions (Mg and Ca) on Cr(VI) reduction with organic reducing substances in the form of sugarcane molasses at different conditions. Results illustrated that the reaction could be described as pseudo-first-order with respect to the Cr(VI) concentration. Reaction rate constants of Cr(VI) reduction with sugarcane molasses were in the decreasing order of no hardness ions > Ca(II) > Mg(II). The possible mechanism was that the complex formation between organic reducing substances and hardness ions (Mg or Ca) hampered the electron transfer rate of the organic reducing substances, and further led to a decrease in the reaction rate. For Mg(II), the initial reaction rates at different pH values (2.0, 2.5, 3.0, and 3.5) decreased by 0.0316, 0.0246, 0.0095, and 0.0023/h in the presence of Mg(II) compared with those in the absence of Mg(II). The inhibitory effect of Mg(II) on the reduction of Cr(VI) was present at different temperatures. The activation energy (Ea) was estimated at 57.4 and 65.6 kJ/mol in the presence and absence of Mg(II), respectively. This would be significant in promoting understanding influence mechanism of hardness ions on Cr(VI) reduction with sugarcane molasses.

Folding behavior of a T-shaped, ribosome-binding translation enhancer implicated in a wide-spread conformational switch.

Turnip crinkle virus contains a T-shaped, ribosome-binding, translation enhancer (TSS) in its 3'UTR that serves as a hub for interactions throughout the region. The viral RNA-dependent RNA polymerase (RdRp) causes the TSS/surrounding region to undergo a conformational shift postulated to inhibit translation. Using optical tweezers (OT) and steered molecular dynamic simulations (SMD), we found that the unusual stability of pseudoknotted element H4a/Ψ3 required five upstream adenylates, and H4a/Ψ3 was necessary for cooperative association of two other hairpins (H5/H4b) in Mg2+. SMD recapitulated the TSS unfolding order in the absence of Mg2+, showed dependence of the resistance to pulling on the 3D orientation and gave structural insights into the measured contour lengths of the TSS structure elements. Adenylate mutations eliminated one-site RdRp binding to the 3'UTR, suggesting that RdRp binding to the adenylates disrupts H4a/Ψ3, leading to loss of H5/H4b interaction and promoting a conformational switch interrupting translation and promoting replication.

In-situ ellipsometric study of calcium phosphate biomineralisation on organic thin films

In this study, in-situ ellipsometry and scanning electron microscopy (SEM) were used to follow the growth kinetics of calcium phosphate (CaP) films on zein and hexadecanoic acid (HDA) layers self-assembled at the air-liquid interface. The duration of the mineral film induction period was heavily dependent on the organic template used, presence or absence of Mg2+, solution pH and concentration. In a standard simulated body fluid (SBF) solution at 20°C and pH 7.4, CaP film growth occurred after an induction period of 40 min for HDA and 100 min for zein. In the absence of Mg2+ the mineralisation induction period was reduced, for only the zein protein, to 50 min. At pH 6, HDA and zein-induced CaP films started forming after 100 min and 180 min, respectively. No induction period was observed when the SBF concentration was doubled, with immediate CaP growth observed for both HDA and zein. This study shows the effect of biomineralisation conditions on the growth of CaP with the organic template playing an important role. Knowledge of the factors that influence mineral growth processes is of importance in areas of biomineralisation and controlled crystal growth.

Novel injectable, self-gelling hydrogel–microparticle composites for bone regeneration consisting of gellan gum and calcium and magnesium carbonate microparticles

The suitability of hydrogel biomaterials for bone regeneration can be improved by incorporation of an inorganic phase in particle form, thus maintaining hydrogel injectability. In this study, carbonate microparticles containing different amounts of calcium (Ca) and magnesium (Mg) were added to solutions of the anionic polysaccharide gellan gum (GG) to crosslink GG by release of Ca2+ and Mg2+ from microparticles and thereby induce formation of hydrogel–microparticle composites. It was hypothesized that increasing Mg content of microparticles would promote GG hydrogel formation. The effect of Mg incorporation on cytocompatibility and cell growth was also studied. Microparticles were formed by mixing Ca2+ and Mg2+ and ions in varying concentrations. Microparticles were characterized physiochemically and subsequently mixed with GG solution to form hydrogel–microparticle composites. The elemental Ca:Mg ratio in the mineral formed was similar to the Ca:Mg ratio of the ions added. In the absence of Mg, vaterite was formed. At low Mg content, magnesian calcite was formed. Increasing the Mg content further caused formation of amorphous mineral. Microparticles of vaterite and magnesium calcite did not induce GG hydrogel formation, but addition of Mg-richer amorphous microparticles induced gelation within 20 min. Microparticles were dispersed homogeneously in hydrogels. MG-63 osteoblast-like cells were cultured in eluate from hydrogel–microparticle composites and on the composites themselves. All composites were cytocompatible. Cell growth was highest on composites containing particles with an equimolar Ca:Mg ratio. In summary, carbonate microparticles containing a sufficient amount of Mg induced GG hydrogel formation, resulting in injectable, cytocompatible hydrogel–microparticle composites.

Magnesium bicarbonate and carbonate interactions in aqueous solutions: An infrared spectroscopic and quantum chemical study

The interaction of magnesium with bicarbonate and carbonate ions in aqueous solutions was studied using infrared spectroscopy and quantum chemical calculations. Using the infrared vibrational bands for HCO3- and CO32- at 1200–1450cm−1 (δC-OH, vS and v3) together with their molar absorptivity (ε), the concentrations of the HCO3- and CO32- ions and the corresponding Mg ion pairs have been determined. In the absence of Mg2+, measured spectra were accurately reproduced assuming that only HCO3- and CO32- were present in solution. Upon addition of Mg2+ at fixed pH, infrared spectra were observed to shift indicating presence of the MgHCO3+ and MgCO3(aq) ion pairs. From measurements, the second ionization constant of carbonic acid and the MgHCO3+ and MgCO3(aq) ion pair formation constants have been obtained, these being logK2=−10.34±0.04, logKMgHCO3+=1.12±0.11 and logKMgCO3=2.98±0.06, respectively. To support our experimental infrared measurements and to gain further insight into the molecular nature of the ion pair formation, density functional theory (DFT) calculations with VPT2 anharmonic correction were conducted. The most stable geometries predicted for the MgHCO3+ and MgCO3(aq) ion pairs were a bi-dentate [MgHCO3]+(H2O)n and a monodentate [MgHCO3]+(OH)(H2O)n complexes, respectively. The predicted frequencies for HCO3-, CO32- and MgHCO3+ were found to shift toward those experimentally measured with an increasing H2O solvation number where possible band shifts were predicted for MgCO3(aq) relative to CO32-, this being dependent on the exact structure and hydration of the bulk MgCO3(aq) ion pair. Experimentally, the ion pair formations were found to have insignificant effects on the δC-OH, vS and v3 vibrational frequencies. The speciation of dissolved inorganic carbon may be significantly influenced by ion pair formation, particularly in alkaline solutions where they may be the predominant species.

Analysis of the Mechanism of Mg2+ Action on the RNase Activity of Serratia marcescens Endonuclease

The main goal of the investigation was to analyze the mechanism of Mg2+ action on the digestive activity of Serratia marcescens nuclease towards RNA due to the potential application of the nuclease as a reagent for removing nucleic acids from biomedical samples as well as an antiviral factor. Examination of the mechanism revealed that it was similar to the mechanism of the metals actions on the DNase activity. The optimal Mg2+ amount was linked with the changing secondary structure of RNA—substrates within A-helix. Addition of Mg2+ was found to affect both the rates of products dissociations from the enzyme-substrate complexes and the enzyme associations with the substrates, that was supported by strong increase in the Kcat values and change in the Km values. Comparing the mechanisms of Mg2+ action on RNase and DNase activities in S. marcescens nuclease, we identified a preference for DNA in the absence of Mg2+ that was supported with the Kcat values.

Fluorescent Protein-Based Ca2+ Sensor Reveals Global, Divalent Cation-Dependent Conformational Changes in Cardiac Troponin C.

Cardiac troponin C (cTnC) is a key effector in cardiac muscle excitation-contraction coupling as the Ca2+ sensing subunit responsible for controlling contraction. In this study, we generated several FRET sensors for divalent cations based on cTnC flanked by a donor fluorescent protein (CFP) and an acceptor fluorescent protein (YFP). The sensors report Ca2+ and Mg2+ binding, and relay global structural information about the structural relationship between cTnC’s N- and C-domains. The sensors were first characterized using end point titrations to decipher the response to Ca2+ binding in the presence or absence of Mg2+. The sensor that exhibited the largest responses in end point titrations, CTV-TnC, (Cerulean, TnC, and Venus) was characterized more extensively. Most of the divalent cation-dependent FRET signal originates from the high affinity C-terminal EF hands. CTV-TnC reconstitutes into skinned fiber preparations indicating proper assembly of troponin complex, with only ~0.2 pCa unit rightward shift of Ca2+-sensitive force development compared to WT-cTnC. Affinity of CTV-TnC for divalent cations is in agreement with known values for WT-cTnC. Analytical ultracentrifugation indicates that CTV-TnC undergoes compaction as divalent cations bind. C-terminal sites induce ion-specific (Ca2+ versus Mg2+) conformational changes in cTnC. Our data also provide support for the presence of additional, non-EF-hand sites on cTnC for Mg2+ binding. In conclusion, we successfully generated a novel FRET-Ca2+ sensor based on full length cTnC with a variety of cellular applications. Our sensor reveals global structural information about cTnC upon divalent cation binding.

Structural effects of modified ribonucleotides and magnesium in transfer RNAs

Modified nucleotides are ubiquitous and important to tRNA structure and function. To understand their effect on tRNA conformation, we performed a series of molecular dynamics simulations on yeast tRNAPhe and tRNAinit, Escherichia coli tRNAinit and HIV tRNALys. Simulations were performed with the wild type modified nucleotides, using the recently developed CHARMM compatible force field parameter set for modified nucleotides (J. Comput. Chem.2016, 37, 896), or with the corresponding unmodified nucleotides, and in the presence or absence of Mg2+. Results showed a stabilizing effect associated with the presence of the modifications and Mg2+ for some important positions, such as modified guanosine in position 37 and dihydrouridines in 16/17 including both structural properties and base interactions. Some other modifications were also found to make subtle contributions to the structural properties of local domains. While we were not able to investigate the effect of adenosine 37 in tRNAinit and limitations were observed in the conformation of E. coli tRNAinit, the presence of the modified nucleotides and of Mg2+ better maintained the structural features and base interactions of the tRNA systems than in their absence indicating the utility of incorporating the modified nucleotides in simulations of tRNA and other RNAs.

The effect of minor solute additions on the precipitation path of an Al[sbnd]Cu[sbnd]Li alloy

Minor solute additions of Mg, Ag or Zn to AlCuLi alloys are known to promote the formation of the T1-Al2CuLi phase and improve the strengthening of these alloys. In this paper, we use atomic resolution high angle annular dark field scanning transmission electron microscopy (STEM-HAADF) in conjunction with electron dispersive spectroscopy (EDS) mapping to elucidate the mechanisms by which these minor solute additions modify the precipitation path. The results show that the presence of Mg profoundly changes the nature of the precipitates formed, as early as the nucleation stage. In the presence of Mg, precursor phases containing Mg and Cu form at the dislocations, and later lead to a microstructure with GPB zones and S phase together with a high number density of T1 precipitates. In the absence of Mg, GPI zones form in the early stages of precipitation and later lead to a microstructure dominated by θ′ precipitates, with a low density of T1. Ag and Zn additions do not change the precipitation sequence, yet they are found in the T1 precipitates, Ag located at the precipitate/matrix interface, and Zn within the precipitate, probably substituting for Cu.

Single-Particle Cryo-EM Studies of a 200-kDa Magnesium Ion Channel Reveal Large Structural Changes Upon Gating

Single-particle cryo-EM was used to explore the structure and conformational landscape of ∼200-kDa magnesium ion channel CorA. CorA is the major Mg2+-uptake system in prokaryotes and can functionally complement the eukaryotic mitochondrial Mrs2 channel, which is essential for normal mitochondrial activity, and whose abnormal expression has been associated with diseases ranging from cancer to demyelination of neuronal tissue. CorA is a homo-pentamer and is gated by intracellular Mg2+. X-ray crystallographic studies of CorA show similar, rather symmetric conformations under Mg2+-bound and Mg2+-free conditions, but EPR spectroscopic studies reveal large Mg2+-driven quaternary conformational changes. Here, we determined the cryo-EM structures of CorA in the presence and absence of Mg2+. The five-fold symmetric Mg2+-bound “closed” state of detergent-solubilized CorA at an overall 3.8-A resolution reveals side-chain densities as well as densities for several magnesium ions, and is similar to known X-ray structures with only minor differences in some of the transmembrane segments, the C-terminal domain, and the periplasmic loops. Cryo-EM of Mg2+-free “open” state CorA shows dramatic differences in the conformation of the channel with large asymmetric changes in the soluble domain of the pentamer. In the absence of bound Mg2+, four of the five subunits are displaced ∼10 to ∼25 A by hinge-like motions at the stalk helix with a bending of up to 35°. Extensive 3D-classification during data-processing resulted in at least two distinct “open” state CorA structures at ∼7-A. The asymmetric structures were supported by single-particle cryo-EM of CorA in lipid nanodiscs and other biochemical and biophysical studies. Our findings suggest an unprecedented gating mechanism where the reversible loss of five-fold symmetry is the key structural signature of the transition between a single “closed” state and an ensemble of low-Mg2+, “open” conformational states.

Biomimetic mineralization of synthetic collagen-like peptide: a bottom-up approach to design advanced nanocomposite scaffolds for tissue engineering

Event Abstract Back to Event Biomimetic mineralization of synthetic collagen-like peptide: a bottom-up approach to design advanced nanocomposite scaffolds for tissue engineering Gloria Belen Ramirez Rodriguez1, José Manuel Delgado-López2, Michele Iafisco1, Gopal Shankar3, Monica Montesi1, Silvia Panseri1, Monica Sandri1, Simone Sprio1, Bas Kluijtmans4 and Anna Tampieri1 1 Institute of Science and Technology for Ceramic, Bioceramics and Bio-hybrid Composites, Italy 2 Instituto Andaluz de Ciencias de la Tierra, IACT-UGR-CSIC, Spain 3 Rizzoli Orthopaedic Institute, Italy 4 FUJIFILM Manufacturing Europe B.V., TRL Life Science, Netherlands Bone extracellular matrix (ECM) consists in self-assembled nanostructure of proteins (mainly type I collagen) and poor crystalline hydroxyapatite nanocrystals (HA) that surrounds and affects mesenchymal stem cell adhesion, proliferation and differentiation through physico-chemical processes[1]. In attempts to orchestrate in vivo bone environment, we propose biomineralization of a synthetic collagen peptide (SCP, manufactured as CellnestTM by FUJIFILM Europe B.V.). Furthermore, inspired by non-stoichiometric composition of bone apatite, magnesium (Mg) is introduced during mineralization process. Mg controls HA crystallization process as well as can stimulate the osteoblast proliferation[2] and inhibit bacterial adhesion[3]. Variation on morphological and chemical features of HA related to the effect of SCP and Mg during mineralization affect surface nanotopography and consequently micro- and macroscopic interfacial scaffold properties with great relevance on cell interaction. Mineralization of synthetic collagen like-peptide (SCP) was carried out by neutralization process[4] in the absence (SCP-HA) and in the presence of magnesium (SCP-MgHA). The effect of SCP and Mg in HA crystallization was evaluated through the morphological characterization by X-Ray powder diffraction (patterns analyzed by Debye Function Analysis[5]) and transmission electron microscopy (TEM). Chemical analysis was performed by Raman spectroscopy, inductively coupled plasma optical emission spectrometry (ICP-OES) and thermogravimetric analysis. Looking ahead, mineralized solution was directly freeze-dried for the development of porous 3D scaffolds. After that, scaffolds were chemical crosslinked to ensure their stability for long-term biomedical applications. Scaffold surface topography was assessed by High-Resolution Atomic Force Microscopy (HR-AFM). The biological response of MC3T3-E1 cell line to scaffolds was assessed, too. Poor crystalline carbonated HA with similar features to that of bone apatites was synthesized. The amorphous calcium phosphate phase (ACP) was present in all conditions being the main phase in SCP-MgHA mineralization experiments which indicated the synergistic effect of SCP and Mg in stabilization of amorphous phase. Moreover, HR-AFM images of scaffold surface showed that mineralization under this condition (SCP-MgHA) lead to homogeneous mineral distribution while non-covered zones together with isolated HA nanocrystals and/or HA aggregates appeared when mineralization is carried out in the absence of Mg (SCP-HA). MC3T3-E1 cells spread inside the material and exhibited their characteristic morphology which could indicate the osteoconductive effects of designed scaffolds. Figure 1. 3D HR-AFM images of non-mineralized and mineralized scaffold surface as well as surface roughness in terms of root mean square (RMS) roughness (Rq) for scan area 2.5µm x 2.5 µm. Figure 2. SEM micrographs of MC3T3-E1 cells attached to non-mineralized and mineralized scaffolds after 7 days of culture. Herein, we propose biomineralization as a versatile route for the development of biomaterials for bone tissue engineering from nano- to macro-scopic scale. The introduction of foreign ions during mineralization allows us to modify physico-chemical properties that cue cell behavior. In our studies, synergistic effect of SCP and Mg on mineralization provided a homogeneous amorphous mineral distribution triggering to a more resorbable biomaterial. In vitro studies showed that cells attached, proliferated and colonized the scaffolds after 7 days. The research leading to these results has received funding from the European Union Seventh Framework Programme FP7-PEOPLE-2013-ITN under grant agreement n 607051

Novel self-gelling, injectable composites for bone regeneration based on gellan gum hydrogel and calcium and magnesium carbonate microparticles

Event Abstract Back to Event Novel self-gelling, injectable composites for bone regeneration based on gellan gum hydrogel and calcium and magnesium carbonate microparticles Timothy Douglas1, Agata Lapa2, Katarzyna Reczynska2, Malgorzata Krok-Borkowicz2, Krzysztof Pietryga2, Sangram K. Samal3, 4, David Schaubroeck5, Marijn Boone6, Pascal Van Der Voort7, Karel De Schamphelaere8, Christian V. Stevens9, Veerle Cnudde6, Elzbieta Pamula2 and Andre G. Skirtach1, 4 1 Ghent University, Molecular Biotechnology, Belgium 2 AGH University of Science and Technology, Biomaterials, Poland 3 Ghent University, General Biochemistry and Physical Pharmacy, Belgium 4 Ghent University, Centre for Nano- and Biophotonics,, Belgium 5 Ghent University, Center for Microsystems Technology (CMST), Belgium 6 Ghent University, Geology and Soil Science, Belgium 7 Ghent University, Inorganic Chemistry, Belgium 8 Ghent University, Laboratory for Environmental and Aquatic Ecology, Environmental Toxicology Unit (GhEnToxLab), Belgium 9 Ghent University, Sustainable Organic Chemistry and Technology, Belgium Introduction: Hydrogels are becoming more popular biomaterials for bone regeneration due to their injectability and the ease of incorporation of inorganic particles, e.g. calcium phosphate (CaP)[1]. Carbonates, e.g. CaCO3 have been successfully applied as bone regeneration materials and are more soluble than CaP. Carbonate particles could potentially serve as delivery vehicles for slow release of calcium (Ca) or magnesium (Mg) ions to crosslink anionic polysaccharides such as gellan gum (GG) to form homogeneous hydrogels. Carbonate microparticles containing Ca and/or Mg can be formed easily by mixing Ca2+ and Mg2+ and CO32- ions in varying concentrations. In this study, carbonate microparticles containing different amounts of Ca and Mg were added to a GG solution. Ca2+ and Mg2+ released from microparticles crosslinked GG to yield hydrogel-microparticle composites. The effect of Mg content on gelation, cytocompatibility and cell growth was studied. Methods: Ca2+/Mg2+ solutions of different Ca:Mg elemental ratios and CO32- solution were prepared. Particles were separated off by centrifugation, dried and autoclaved and subjected to physiochemical characterization using FTIR, XRD, SEM, Raman, AAS and DLS. To generate self-gelling GG-microparticle composites, microparticles were resuspended in ddH2O and mixed with autoclaved GG solution at 37º C. Final microparticle and GG concentrations were 2.5% and 0.66%, respectively (w/v). Gelation speed, microparticle distribution and cytocompatibility were investigated by rheometry, Micro-CT and MG-63 osteoblast-like cell culture in eluate from composites and directly on composites, respectively. Results: In the absence of Mg, calcite with some vatertie was formed. At low Mg content, magnesian calcite was formed. Increasing Mg content further increased general amorphicity. Microparticles of calcite, vaterite and magnesium calcite did not induce hydrogel formation. Addition of Mg-richer microparticles induced gelation within 25 min. Microparticle dispersion in hydrogels was homogeneous. All composites were cytocompatible. Cell growth after 7 d was highest on composites containing particles with an equimolar Ca:Mg ratio. Discussion: Increasing Mg content in the microparticles increased their amorphicity, in turn increasing their solubility, resulting in enhanced ion release and GG crosslinking and, in turn, hydrogel formation. Other studies have reported a beneficial effect of Mg as a component of CaP on cell proliferation[2], as observed in this study. Conclusion: Carbonate microparticles containing a sufficient amount of Mg induce GG hydrogel formation, resulting in injectable, cytocompatible hydrogel-microparticle composites. FWO, Belgium

Aligned deposition and electrical measurements on single DNA molecules

A reliable method of deposition of aligned individual dsDNA molecules on mica, silicon, and micro/nanofabricated circuits is presented. Complexes of biotinylated double stranded poly(dG)–poly(dC) DNA with avidin were prepared and deposited on mica and silicon surfaces in the absence of Mg2+ ions. Due to its positive charge, the avidin attached to one end of the DNA anchors the complex to negatively charged substrates. Subsequent drying with a directional gas flow yields DNA molecules perfectly aligned on the surface. In the avidin–DNA complex only the avidin moiety is strongly and irreversibly bound to the surface, while the DNA counterpart interacts with the substrates much more weakly and can be lifted from the surface and realigned in any direction. Using this technique, avidin–DNA complexes were deposited across platinum electrodes on a silicon substrate. Electrical measurements on the deposited DNA molecules revealed linear IV-characteristics and exponential dependence on relative humidity.

Mg(2+) shifts ligand-mediated folding of a riboswitch from induced-fit to conformational selection.

Bacterial riboswitches couple small-molecule ligand binding to RNA conformational changes that widely regulate gene expression, rendering them potential targets for antibiotic intervention. Despite structural insights, the ligand-mediated folding mechanisms of riboswitches are still poorly understood. Using single-molecule fluorescence resonance energy transfer (smFRET), we have investigated the folding mechanism of an H-type pseudoknotted preQ1 riboswitch in dependence of Mg(2+) and three ligands of distinct affinities. We show that, in the absence of Mg(2+), both weakly and strongly bound ligands promote pseudoknot docking through an induced-fit mechanism. By contrast, addition of as low as 10 μM Mg(2+) generally shifts docking toward conformational selection by stabilizing a folded-like conformation prior to ligand binding. Supporting evidence from transition-state analysis further highlights the particular importance of stacking interactions during induced-fit and of specific hydrogen bonds during conformational selection. Our mechanistic dissection provides unprecedented insights into the intricate synergy between ligand- and Mg(2+)-mediated RNA folding.

Assessment of metal-assisted nucleophile activation in the hepatitis delta virus ribozyme from molecular simulation and 3D-RISM.

The hepatitis delta virus ribozyme is an efficient catalyst of RNA 2′-O-transphosphorylation and has emerged as a key experimental system for identifying and characterizing fundamental features of RNA catalysis. Recent structural and biochemical data have led to a proposed mechanistic model whereby an active site Mg2+ ion facilitates deprotonation of the O2′ nucleophile, and a protonated cytosine residue (C75) acts as an acid to donate a proton to the O5′ leaving group as noted in a previous study. This model assumes that the active site Mg2+ ion forms an inner-sphere coordination with the O2′ nucleophile and a nonbridging oxygen of the scissile phosphate. These contacts, however, are not fully resolved in the crystal structure, and biochemical data are not able to unambiguously exclude other mechanistic models. In order to explore the feasibility of this model, we exhaustively mapped the free energy surfaces with different active site ion occupancies via quantum mechanical/molecular mechanical (QM/MM) simulations. We further incorporate a three-dimensional reference interaction site model for the solvated ion atmosphere that allows these calculations to consider not only the rate associated with the chemical steps, but also the probability of observing the system in the presumed active state with the Mg2+ ion bound. The QM/MM results predict that a pathway involving metal-assisted nucleophile activation is feasible based on the rate-controlling transition state barrier departing from the presumed metal-bound active state. However, QM/MM results for a similar pathway in the absence of Mg2+ are not consistent with experimental data, suggesting that a structural model in which the crystallographically determined Mg2+ is simply replaced with Na+ is likely incorrect. It should be emphasized, however, that these results hinge upon the assumption of the validity of the presumed Mg2+-bound starting state, which has not yet been definitively verified experimentally, nor explored in depth computationally. Thus, further experimental and theoretical study is needed such that a consensus view of the catalytic mechanism emerges.

The Lost City hydrothermal system: Constraints imposed by vent fluid chemistry and reaction path models on subseafloor heat and mass transfer processes

Since the first reported discovery of the Lost City hydrothermal system in 2001, it was recognized that seawater alteration of ultramafic rocks plays a key role in the composition of the coexisting vent fluids. The unusually high pH and high concentrations of H2 and CH4 provide compelling evidence for this. Here we report the chemistry of hydrothermal fluids sampled from two vent structures (Beehive: ∼90–116°C, and M6: ∼75°C) at Lost City in 2008 during cruise KNOX18RR using ROV Jason 2 and R/V Revelle assets. The vent fluid chemistry at both sites reveals considerable overlap in concentrations of dissolved gases (H2, CH4), trace elements (Cs, Rb, Li, B and Sr), and major elements (SO4, Ca, K, Na, Cl), including a surprising decrease in dissolved Cl, suggesting a common source fluid is feeding both sites. The absence of Mg and relatively high concentrations of Ca and sulfate suggest solubility control by serpentine–diopside–anhydrite, while trace alkali concentrations, especially Rb and Cs, are high, assuming a depleted mantle protolith. In both cases, but especially for Beehive vent fluid, the silica concentrations are well in excess of those expected for peridotite alteration and the coexistence of serpentine–brucite at all reasonable temperatures. However, both the measured pH and silica values are in better agreement with serpentine–diopside–tremolite-equilibria. Geochemical modeling demonstrates that reaction of plagioclase with serpentinized peridotite can shift the chemical system away from brucite and into the tremolite stability field. This is consistent with the complex intermingling of peridotite and gabbroic bodies commonly observed within the Atlantis Massif. We speculate the existence of such plagioclase bearing peridotite may also account for the highly enriched trace alkali (Cs, Rb) concentrations in the Lost City vent fluids. Additionally, reactive transport modeling taking explicit account of temperature dependent rates of mineral dissolution and precipitation clarifies the feedback between permeability, heat loss, and changes in the dissolved Si of the vent fluids. Assuming both the Beehive and M6 vent fluids were sourced at similar subseafloor conditions (tremolite buffered at 200°C), model results indicate loss of approximately 30% Si upon cooling to ∼150°C during upflow. However, Si concentrations remained largely conservative with continued cooling to lower temperatures owing to unfavorable reaction kinetics. While consistent with the Beehive endmember composition, these results fail to explain the relative Si depletion in the lower temperature M6 fluids. Thus, it may be that more robust kinetic models for silicates are needed to accurately account for the mechanism and rate of silica removal in the unusually high pH of the Lost City vent fluids.

Inverse thio effects in the hepatitis delta virus ribozyme reveal that the reaction pathway is controlled by metal ion charge density.

The hepatitis delta virus (HDV) ribozyme self-cleaves in the presence of a wide range of monovalent and divalent ions. Prior theoretical studies provided evidence that self-cleavage proceeds via a concerted or stepwise pathway, with the outcome dictated by the valency of the metal ion. In the present study, we measure stereospecific thio effects at the nonbridging oxygens of the scissile phosphate under a wide range of experimental conditions, including varying concentrations of diverse monovalent and divalent ions, and combine these with quantum mechanical/molecular mechanical (QM/MM) free energy simulations on the stereospecific thio substrates. The RP substrate gives large normal thio effects in the presence of all monovalent ions. The SP substrate also gives normal or no thio effects, but only for smaller monovalent and divalent cations, such as Li+, Mg2+, Ca2+, and Sr2+; in contrast, sizable inverse thio effects are found for larger monovalent and divalent cations, including Na+, K+, NH4+, and Ba2+. Proton inventories are found to be unity in the presence of the larger monovalent and divalent ions, but two in the presence of Mg2+. Additionally, rate–pH profiles are inverted for the low charge density ions, and only imidazole plus ammonium ions rescue an inactive C75Δ variant in the absence of Mg2+. Results from the thio effect experiments, rate–pH profiles, proton inventories, and ammonium/imidazole rescue experiments, combined with QM/MM free energy simulations, support a change in the mechanism of HDV ribozyme self-cleavage from concerted and metal ion-stabilized to stepwise and proton transfer-stabilized as the charge density of the metal ion decreases.

Learning from ribozymes

Learning from ribozymes