The role of protein–solvent hydrogen bond dynamics in the structural relaxation of a protein in glycerol versus water

We used MD simulations to investigate the dependence of the dynamics of a soluble protein, RNase A, on temperature and solvent environment. Consistent with neutron scattering data, the simulations predict that the protein undergoes a dynamical transition in both glycerol and aqueous solutions that is absent in the dry protein. The temperature of the transition is higher, while the rate of increase with temperature of the amplitudes of motion on the 100 ps timescale is lower, in glycerol versus water. Analysis of the dynamics of hydrogen bonds revealed that the protein dynamical transition is connected to the relaxation of the protein-solvent hydrogen bond network, which, in turn, is associated with solvent translational diffusion. Thus, it appears that the role of solvent dynamics in affecting the protein dynamical transition is qualitatively similar in water and glycerol.     

Presence of Ornithine in the Urate-binding α<Subscript>1</Subscript>—<Subscript>2</Subscript>Globulin

THE urate-binding α₁–α₂ globulin has been isolated from human plasma in a highly purified state¹. The protein was purified by DEAE-‘Sephadex’, ammonium sulphate precipitation and semi-preparative Polyacrylamide gel electrophoresis. The urate-binding α₁–α₂ globulin is a rod-shaped glycoprotein, containing 12.1% carbohydrate, with an isoelectric point of 4.6 and a molecular weight of 67,000 ± 4,000. Amino-acid analysis indicated an unknown basic compound which appeared as an extra peak just in front of lysine¹. To identify this compound, high voltage paper electrophoresis has been carried out on a plate electrophoresis apparatus in pyridine-acetate buffer pH 3.5. A spot separated out corresponding to ornithine. Amino-acid analysis on a BC-200 automatic analyser (Bio-Cal Instruments Co., West Germany), with a 54 cm column at 55° C and with 0.35 M sodium citrate buffer, pH 5.28, as elution buffer at a flow-rate of 150 ml./h, showed that ornithine was present. The presence of ornithine in the protein hydrolysate was also verified by gas chromatography/mass spectrometry².     

Hydrogen-bonding behavior of various conformations of the HNO<Subscript>3</Subscript>…(CH<Subscript>3</Subscript>OH)<Subscript>2</Subscript> ternary system

Nine minima were found on the intermolecular potential energy surface for the ternary system HNO₃(CH₃OH)₂ at the MP2/aug-cc-pVDZ level of theory. The cooperative effect, which is a measure of the hydrogen-bonding strength, was probed in these nine conformations of HNO₃…(CH₃OH)₂. The results are discussed here in terms of structures, energetics, infrared vibrational frequencies, and topological parameters. The cooperative effect was observed to be an important contributor to the total interaction energies of the cyclic conformers of HNO₃…(CH₃OH)₂, meaning that it cannot be neglected in simulations in which the pair-additive potential is applied.

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Graphical abstract The H-bonding behavior of various conformations of the HNO₃(CH₃OH)₂ trimer was investigated

     

The neural γ<Subscript>2</Subscript>α<Subscript>1</Subscript>β<Subscript>2</Subscript>α<Subscript>1</Subscript>β<Subscript>2</Subscript> gamma amino butyric acid ion channel receptor: structural analysis of the effects of the ivermectin molecule and disulfide bridges

While ~30% of the human genome encodes membrane proteins, only a handful of structures of membrane proteins have been resolved to high resolution. Here, we studied the structure of a member of the Cys-loop ligand gated ion channel protein superfamily of receptors, human type A γ₂α₁β₂α₁β₂ gamma amino butyric acid receptor complex in a lipid bilayer environment. Studying the correlation between the structure and function of the gamma amino butyric acid receptor may enhance our understanding of the molecular basis of ion channel dysfunctions linked with epilepsy, ataxia, migraine, schizophrenia and other neurodegenerative diseases. The structure of human γ₂α₁β₂α₁β₂ has been modeled based on the X-ray structure of the Caenorhabditis elegans glutamate-gated chloride channel via homology modeling. The template provided the first inhibitory channel structure for the Cys-loop superfamily of ligand-gated ion channels. The only available template structure before this glutamate-gated chloride channel was a cation selective channel which had very low sequence identity with gamma aminobutyric acid receptor. Here, our aim was to study the effect of structural corrections originating from modeling on a more reliable template structure. The homology model was analyzed for structural properties via a 100 ns molecular dynamics (MD) study. Due to the structural shifts and the removal of an open channel potentiator molecule, ivermectin, from the template structure, helical packing changes were observed in the transmembrane segment. Namely removal of ivermectin molecule caused a closure around the Leu 9 position along the ion channel. In terms of the structural shifts, there are three potential disulfide bridges between the M1 and M3 helices of the γ₂ and 2 α₁ subunits in the model. The effect of these disulfide bridges was investigated via monitoring the differences in root mean square fluctuations (RMSF) of individual amino acids and principal component analysis of the MD trajectory of the two homology models—one with the disulfide bridge and one with protonated Cys residues. In all subunit types, RMSF of the transmembrane domain helices are reduced in the presence of disulfide bridges. Additionally, loop A, loop F and loop C fluctuations were affected in the extracellular domain. In cross-correlation analysis of the trajectory, the two model structures displayed different coupling in between the M2–M3 linker region, protruding from the membrane, and the β1-β2/D loop and cys-loop regions in the extracellular domain. Correlations of the C loop, which collapses directly over the bound ligand molecule, were also affected by differences in the packing of transmembrane helices. Finally, more localized correlations were observed in the transmembrane helices when disulfide bridges were present in the model. The differences observed in this study suggest that dynamic coupling at the interface of extracellular and ion channel domains differs from the coupling introduced by disulfide bridges in the transmembrane region. We hope that this hypothesis will be tested experimentally in the near future.     

Catalytic mechanisms of Au<Subscript>11</Subscript> and Au<Subscript>11-n</Subscript>Pt<Subscript>n</Subscript> (<Emphasis Type="Italic">n</Emphasis>=1–2) clusters: a DFT investigation on the oxidation of CO by O<Subscript>2</Subscript>

The oxidation of CO catalyzed by clusters of Au₁₁, Au₁₀Pt and Au₉Pt₂ was investigated using the M06 functional suite of the density functional theory. Au and Pt atoms were described with the double-ζ valence basis set Los Alamos National Laboratory 2-double-z (LanL2DZ), whereas the standard 6-311++G(d,p) basis set was employed for the C and O atoms. Our theoretical model showed that (1) after coordination to Au and Au-Pt cluster, O₂ and CO are apparently activated, and Mulliken charges show that the gold atoms in the active sites of Au11 are negatively charged; (2) Au-Pt clusters with 11 atoms can effectively catalyze the oxidation of CO by O₂; (3) Au₁₁ exhibits good catalytic performance for the oxidation of CO; (4) oxidation of CO occurs preferably on the Au–Pt active sites in Pt-doped clusters, and the single-center mechanisms are more favorable energetically than the two-center mechanisms; (5) after adsorption, an O₂ molecule oxidates two CO molecules via stepwise mechanisms; and (6) the catalytic processes are highly exothermic.     

The Release of CO<Subscript>2</Subscript> from Riverwaters – the Contribution of Excess CO<Subscript>2</Subscript> from Groundwater

The dissolved CO ₂ concentration of stream waters is an important component of the terrestrial carbon cycle and an important pathway for release of CO₂ to the atmosphere. This study uses data from the UK's largest groundwater monitoring network to estimate the importance of groundwater in contributing excess dissolved CO₂ to the atmosphere. The study shows that:

The effect of metal ions on the binding of ethanol to human alcohol dehydrogenase β<Subscript>2</Subscript>β<Subscript>2</Subscript>

Molecular docking simulations were performed in this study to investigate the importance of both structural and catalytic zinc ions in the human alcohol dehydrogenase beta(2)beta(2) on substrate binding. The structural zinc ion is not only important in maintaining the structural integrity of the enzyme, but also plays an important role in determining substrate binding. The replacement of the catalytic zinc ion or both catalytic and structural zinc ions with Cu(2+) results in better substrate binding affinity than with the wild-type enzyme. The width of the bottleneck formed by L116 and V294 in the substrate binding pocket plays an important role for substrate entrance. In addition, unfavorable contacts between the substrate and T48 and F93 prevent the substrate from moving too close to the metal ion. The optimal binding position occurs between 1.9 and 2.4 A from the catalytic metal ion.     

Implication of α<Subscript>5</Subscript>β<Subscript>1</Subscript> integrin in invasion of drug-resistant MCF-7/ADR breast carcinoma cells: a role for MMP-2 collagenase

Expression of alpha5beta1 integrin in the drug-resistant MCF-7/ADR breast carcinoma cells was inhibited by treatment of these cells with alpha5-specific siRNA. The decrease of alpha5beta1 expression resulted in a sharp decrease of expression of MMP-2 collagenase and inhibition of invasion activity of these cells in vitro. Similar decrease of invasion was also observed during inhibition of MMP-2 expression by treatment of these cells with MMP-2-specific siRNA. Inhibition of alpha5beta1 expression was also accompanied by significant decrease in cell content of active (phosphorylated) forms of signal protein kinases Akt and Erk1/2. Inhibition of activity of these kinases by treatment of cells with PI-3K/Akt-specific inhibitor LY294002 or Erk-specific inhibitor PD98059 resulted in inhibition of MMP-2 expression and the decrease of invasion in vitro. These data suggest that alpha5beta1 controls invasion ability of these cells by regulating expression of MMP-2, which involves PI-3K and Erk1/2 protein kinase signaling.     

On the generation of OH<Superscript>·</Superscript> radical species from H<Subscript>2</Subscript>O<Subscript>2</Subscript> by Cu(I) amyloid beta peptide model complexes: a DFT investigation

According to different studies, the interaction between amyloid β-peptide (Aβ) and copper ions could yield radical oxygen species production, in particular the highly toxic hydroxyl radical OH^· that is suspected to contribute to Alzheimer’s disease pathogenesis. Despite intensive experimental and computational studies, the nature of the interaction between copper and Aβ peptide, as well as the redox reactivity of the system, are still matter of debate. It was proposed that in Cu(II) → Cu(I) reduction the complex Cu(II)–Aβ could follow a multi-step conformational change with redox active intermediates that may be responsible for OH^· radical production from H₂O₂ through a Fenton-like process. The purpose of this work is to evaluate, using ab initio Density Functional Theory computations, the reactivity of different Cu(I)–Aβ coordination modes proposed in the literature, in terms of OH^· production. For each coordination model, we considered the corresponding H₂O₂ adduct and performed a potential energy surface scan along the reaction coordinate of O–O bond dissociation of the peroxide, resulting in the production of OH^· radical, obtaining reaction profiles for the evaluation of the energetic of the process. This procedure allowed us to confirm the hypothesis according to which the most populated Cu(I)–Aβ two-histidine coordination is not able to perform efficiently H₂O₂ reduction, while a less populated three-coordinated form would be responsible for the OH^· production. We show that coordination modes featuring a third nitrogen containing electron-donor ligand (an imidazole ring of an histidine residue is slightly favored over the N-terminal amine group) are more active towards H₂O₂ reduction.     

An optical material for the detection of trace S<Subscript>2</Subscript>O<Subscript>3</Subscript><Superscript>2−</Superscript> in milk based on a copper complex

A novel S₂O₃ ^2? luminescent sensor (Cu²⁺-p-CPIP) was developed and the presence of S₂O₃ ^2? caused an obvious fluorescence enhancement at 420 nm upon excitation at 330 nm, which could be distinguished with the naked eye under a UV lamp. Remarkably, the compound exhibited excellent selective and sensitive response to S₂O₃ ^2? over other common anions with a micromolar limit of detection (0.442 μM) in DMSO/H₂O (v/v, 1:1) buffer. The absorbance intensity and the color of Cu²⁺-p -CPIP solution changed gradually with the increase of S₂O₃ ^2? concentration. The proposed method was applied to the determination of S₂O₃ ^2? in milk samples and the recoveries were 97.5–105%. The preparation of Cu²⁺-p -CPIP exhibited the quick, simple and facile advantages. The results showed that Cu²⁺-p -CPIP can be a good candidate for simple, rapid and sensitive colorimetric detection of S₂O₃ ^2? in aqueous solution.     

Charge ordering in the metal–insulator transition of V-doped CrO<Subscript>2</Subscript> in the rutile structure

Electronic, magnetic, and structural properties of pure and V-doped CrO₂ were extensively investigated utilizing density functional theory. Usually, pure CrO₂ is a half-metallic ferromagnet with conductive spin majority species and insulating spin minority species. This system remains in its half-metallic ferromagnetic phase even at 50% V-substitution for Cr within the crystal. The V-substituted compound Cr_0.5V_0.5O₂ encounters metal–insulator transition upon the application of on-site Coulomb repulsion U?=?7 eV preserving its ferromagnetism in the insulating phase. It is revealed in this study that Cr³⁺-V⁵⁺ charge ordering accompanied by the transfer of the single V-3d electron to the Cr-3dt_2g orbitals triggers metal–insulator transition in Cr_0.5V_0.5O₂. The ferromagnetism of Cr_0.5V_0.5O₂ in the insulating phase arises predominantly due to strong Hund’s coupling between the occupied electrons in the Cr-t_2g states. Besides this, the ferromagnetic Curie temperature (T_c) decreases significantly due to V-substitution. Interestingly, a structural distortion is observed due to tilting of CrO₆ or VO₆ octahedra across the metal–insulator transition of Cr_0.5V_0.5O₂.

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Graphical abstract The V-doped compound Cr_0.5V_0.5O₂ is found a half-metallic ferromagnet (HMF) in the absence of on-site Coulomb interaction (U). This HMF behavor maintains up to U?=?6 eV. Eventually, this system encounters metal-insulator transition (MIT) upon the application of U?=?7 eV with a band gap of E_g ~ 0.31 eV. Nevertheless, applications of higher U widen the band gaps. In this figure, calculated total (black), Cr-3d (red), V-3d (violet), and O-2p (blue) DOS of Cr_0.5V_0.5O₂ for U?=?8 eV are illustrated. The system is insulating with a band gap of E_g ~ 0.7 eV.

     

Do increasingly depleted δ<Superscript>15</Superscript>N values of atmospheric N<Subscript>2</Subscript>O indicate a decline in soil N<Subscript>2</Subscript>O reduction?

Growing concentrations of N₂O within the atmosphere have been accompanied by decreasing δ¹⁵N values, provoking the hypothesis of a global decline in the rate of N₂O reduction relative to its production in soil. We estimate that the ratio of N₂O produced to N₂O reduced within the soil profile has declined by about 10–25% relative to its pre-industrial value. To a smaller extent, a reduction in the uptake of atmospheric N₂O at the soil surface relative to its emission could also have contributed to the reported isotopic signal. This calls for a greater consideration of the process of N₂O reduction in soil and its role in the global turnover of N₂O.     

Effects of Prostaglandins F<Subscript>2α</Subscript>, E<Subscript>2</Subscript> and E<Subscript>1</Subscript> on Fertility in Mice

PROSTAGLANDIN (PG) F_2αhas antifertility effects in many species^1–3 but there are conflicting suggestions as to its mechanism of action. For example, it may cause the degeneration of the corpus luteum by decreasing blood flow in the uteroovarian vein⁴; alternatively, its action may be due to a hypersecretion of luteinizing hormone (LH) by the pituitary^3,5. I have investigated the effects of PGF_2α, E₂ and E₁ on pregnancy in mice and examined the mechanism of action of PGF_2α.     

Association of β<Subscript>2</Subscript>-microglobulin with the α<Subscript>3</Subscript> domain of H-2D<Superscript>b</Superscript> heavy chain

MHC class I molecules are heterotrimeric complexes composed of heavy chain, ₂-microglobulin (₂m) and short peptide. This trimeric complex is generated in the endoplasmic reticulum (ER), where a peptide loading complex (PLC) facilitates transport from the cytosol and binding of the peptide to the preassembled ER resident heavy chain/₂m dimers. Association of mouse MHC class I heavy chain with ₂m is characterized by allelic differences in the number and/or positions of amino acid interactions. It is unclear, however, whether all alleles follow common binding patterns with minimal contributions by allele-specific contacts, or whether essential contacts with ₂m are different for each allele. While searching for the PLC binding site in the ₃ domain of the mouse MHC class I molecule H-2D^b, we unexpectedly discovered a site critical for binding mouse, but not human, ₂m. Interestingly, amino acids in the corresponding region of another MHC class I heavy chain allele do not make contacts with the mouse ₂m. Thus, there are allelic differences in the modes of binding of ₂m to the heavy chain of MHC class I.     

A DFT study of the catalytic pyrolysis of benzaldehyde on ZnO, γ-Al<Subscript>2</Subscript>O<Subscript>3</Subscript>, and CaO models

The catalytic pyrolysis pathways of carbonyl compounds in coal were systematically studied using density functional theory (DFT), with benzaldehyde (C₆H₅CHO) employed as a coal-based model compound and ZnO, γ-Al₂O₃, and CaO as catalysts. The results show that the products of both pyrolysis and catalytic pyrolysis are C₆H₆ and CO. However, the presence of any of the catalysts changes the reaction pathway and reduces the energy barrier, indicating that these catalysts promote C₆H₅CHO decomposition.

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Graphical abstract The presence of catalysts changes the reaction pathway and the energy barrier decreases in the order Ea (no catalyst)> Ea (CaO)> Ea (γ-Al₂O₃)> Ea (ZnO), indicating that these catalysts promote C₆H₅CHO decomposition.

     

Effect of the β-amyloid peptide Aβ<Subscript>25–35</Subscript> and fullerene C<Subscript>60</Subscript> on the activity of enzymes in erythrocytes

The effect of the β-amyloid peptide Aβ_25–35 and fullerene C₆₀ on the activity of the cytoplasmic enzymes lactate dehydrogenase (LDH) and glutathione peroxidase (GLP), and membrane-bound phosphofructokinase (PFK) and Na⁺,K⁺-ATPase in human erythrocytes has been studied. When used in combination, the cytotoxins decrease the activity of LDH and PFK in a nonadditive manner; in this case, Aβ_25–35 protects PFK against the inhibitory effect of C₆₀. The activity of LDH, GLP, and PFK decreases within the first 2–20 min of incubation of erythrocytes with Aβ_25–35 in the absence of glucose. The addition of glucose sharply decreases the inhibitory action of Aβ_25–35 on LDH and GLP but does not affect the fourfold decrease in activity of PFK; the activity of membrane-bound Na⁺,K⁺-ATPase does not depend on the presence of glucose. Possible mechanisms of interaction of Aβ_25–35 and fullerene C₆₀ with the erythrocyte membrane and enzymes are discussed.     

Correlated motions of C′–N and C<Subscript>α</Subscript>–C<Subscript>β</Subscript> pairs in protonated and per-deuterated GB3

We investigated correlated µs-ms time scale motions of neighboring ¹³C′–¹⁵N and ¹³C_α–¹³C_β nuclei in both protonated and perdeuterated samples of GB3. The techniques employed, NMR relaxation due to cross-correlated chemical shift modulations, specifically target concerted changes in the isotropic chemical shifts of the two nuclei associated with spatial fluctuations. Field-dependence of the relaxation rates permits identification of the parameters defining the chemical exchange rate constant under the assumption of a two-site exchange. The time scale of motions falls into the intermediate to fast regime (with respect to the chemical shift time scale, 100–400 s^?1 range) for the ¹³C′–¹⁵N pairs and into the slow to intermediate regime for the ¹³C_α–¹³C_β pairs (about 150 s^?1). Comparison of the results obtained for protonated and deuterated GB3 suggests that deuteration has a tendency to reduce these slow scale correlated motions, especially for the ¹³C_α–¹³C_β pairs.     

Estimation of denitrification potential in a karst aquifer using the <Superscript>15</Superscript>N and <Superscript>18</Superscript>O isotopes of NO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack>

A confined aquifer in the Malm Karst of the Franconian Alb, South Germany was investigated in order to understand the role of the vadose zone in denitrifiaction processes. The concentrations of chemical tracers Sr²⁺ and Cl^– and concentrations of stable isotope ¹⁸O were measured in spring water and precipitation during storm events. Based on these measurements a conceptual model for runoff was constructed. The results indicate that pre-event water, already stored in the system at the beginning of the event, flows downslope on vertical and lateral preferential flow paths. Chemical tracers used in a mixing model for hydrograph separation have shown that the pre-event water contribution is up to 30%. Applying this information to a conceptual runoff generation model, the values of ¹⁵N and ¹⁸O in nitrate could be calculated. Field observations showed the occurence of significant microbial denitrification processes above the soil/bedrock interface before nitrate percolates through to the deeper horizon of the vadose zone. The source of nitrate could be determined and denitrification processes were calculated. Assuming that the nitrate reduction follows a Rayleigh process one could approximate a nitrate input concentration of about 170 mg/l and a residual nitrate concentration of only about 15%. The results of the chemical and isotopic tracers postulate fertilizers as nitrate source with some influence of atmospheric nitrate. The combined application of hydrograph separation and determination of isotope values in ¹⁵N and ¹⁸O of nitrate lead to an improved understanding of microbial processes (nitrification, denitrification) in dynamic systems.     

Cooperation of adenosine and prostaglandin E<Subscript>2</Subscript> (PGE<Subscript>2</Subscript>) in amplification of cAMP–PKA signaling and immunosuppression

INTRODUCTION: We hypothesize that adenosine and PGE(2) could have a complementary immunosuppressive effect that is mediated via common cAMP-PKA signaling. MATERIALS AND METHODS: To test this hypothesis, the effect of adenosine and PGE(2) on the cytotoxic activity and cytokine production of lymphokine activated killer (LAK) cells was investigated. RESULTS: PGE(2) and adenosine inhibited LAK cells cytotoxic activity and production of INF-gamma, GM-CSF and TNF-alpha. In combination they showed substantially higher inhibition than each modality used alone. Using agonists and antagonists specific for PGE(2) and adenosine receptors we found that cooperation of PGE(2) and adenosine in their inhibitory effects are mediated via EP(2) and A(2A) receptors, respectively. LAK cells have 35-fold higher expression of EP(2) than A(2A). Combined PGE(2) and adenosine treatment resulted in augmentation of cAMP production, PKA activity, CREB phosphorylation and inhibition of Akt phosphorylation. Wortmannin and LY294002 enhanced the suppressive effects of adenosine and PGE(2). In contrast, Rp-8-Br-cAMPS, an inhibitor of PKA type I blocked their immunosuppressive effects, suggesting that the inhibitory effects of PGE(2) and adenosine are mediated via common pathway with activation of cAMP-PKA and inhibition of Akt. CONCLUSION: In comparison to other immunosuppressive molecules (TGF-beta and IL-10), adenosine and PGE(2) are unique in their ability to inhibit the executive function of highly cytotoxic cells. High intratumor levels of adenosine and PGE(2) could protect tumor from immune-mediated destruction by inactivation of the tumor infiltrating functionally active immune cells.