Probing the flexibility of the DsbA oxidoreductase from Vibrio cholerae - a 15N -1H heteronuclear NMR relaxation analysis of oxidised and reduced forms of DsbA

We have determined the structure of the reduced form of the DsbA oxidoreductase from Vibrio cholerae. The reduced structure shows a high level of similarity to the crystal structure of the oxidized form and is typical of this class of enzyme containing a thioredoxin domain with an inserted α-helical domain. Proteolytic and thermal stability measurements show that the reduced form of DsbA is considerably more stable than the oxidized form. NMR relaxation data have been collected and analyzed using a model-free approach to probe the dynamics of the reduced and oxidized states of DsbA. Akaike's information criteria have been applied both in the selection of the model-free models and the diffusion tensors that describe the global motions of each redox form. Analysis of the dynamics reveals that the oxidized protein shows increased disorder on the pico- to nanosecond and micro- to millisecond timescale. Many significant changes in dynamics are located either close to the active site or at the insertion points between the domains. In addition, analysis of the diffusion data shows there is a clear difference in the degree of interdomain movement between oxidized and reduced DsbA with the oxidized form being the more rigid. Principal components analysis has been employed to indicate possible concerted movements in the DsbA structure, which suggests that the modeled interdomain motions affect the catalytic cleft of the enzyme. Taken together, these data provide compelling evidence of a role for dynamics in the catalytic cycle of DsbA.

Effect of structure relaxation on the plastic deformation behaviour in a Zr-based BMG under indenter

Vickers and nano indentations were performed on a structurally relaxed Zr₄₁Ti₁₄Cu_12.5Ni₁₀Be_22.5 bulk metallic glass (BMG), and the evolution of the shear bands in the relaxed BMG was investigated and compared to that in the as-cast alloy. Results indicate that the plastic deformation in the BMG with structure relaxation is accommodated by the semicircular (primary) and radial (secondary) as well as tertiary shear bands. Quantitatively, the shear band density in the relaxed alloy was much lower than that in the as-cast alloy. The annihilation of free volume caused by the annealing was responsible for the embrittlement of the sample with structure relaxation.

A 7Li and 19F NMR relaxation study of LiCF3SO3 in plasticised solid polyether electrolytes

⁷Li and ¹⁹F NMR relaxation time (T₁, T₂, T_1ρ) measurements have been used to probe the dynamics of LiCF₃SO₃ dissolved in an amorphous co-polymer poly(ethylene oxide-co-propylene oxide), and in particular the influence of the plasticising agents propylene carbonate and dimethyl formamide. The changes in relaxation behaviour of ¹⁹F and ⁷Li with increasing plasticiser concentration are very different, as is the effect of each plasticiser. These differences can be explained qualitatively in terms of the interaction between the plasticiser and the ions. Preliminary ⁷Li T_1ρ measurements reveal two components at low temperatures.

The effect of solvent uptake on the relaxation behaviour, morphology and mechanical properties of a poly(ether ether ketone)/poly(etherimide) blend

The effects of solvent uptake on the relaxation behaviour, morphology and mechanical properties of poly(ether ether ketone) (PEEK), poly(etherimide) (PEI) and a 50/50 PEEK/PEI blend have been investigated. Amorphous films were immersed in acetone at 25°C, 35°C and 45°C until equilibrium uptake was achieved. The films were then examined by wide angle X-ray scattering (WAXS), differential scanning calorimetry (d.s.c.), dynamic mechanical relaxation spectroscopy and mechanical testing. WAXS and d.s.c. revealed that the degree of solvent induced crystallinity in PEEK is constant with immersion temperature, whereas the degree of induced crystallinity in the 50/50 blend is strongly temperature dependent. The dynamic mechanical studies confirmed that a significant decrease in glass transition temperature results from the plasticizing effect of the solvent and that solvent and thermally crystallized samples have different relaxation characteristics. Mechanical property tests showed that the yield stress and tensile strength of the blend are dominated by PEEK and the degree of crystallinity, while the modulus is more sensitive to the extent of plasticization.

A study of hydrogen bonding in concentrated diol/water solutions by proton NMR correlations with glass formation

Solute/water interactions in a series of diol solutions have been investigated by ¹H NMR. Strong hydrogen bonding between water and alcohols that are more basic than water is thought to result in lower chemical shifts of water protons compared to the case of pure water. This is attributed to a greater degree of covalent character in the hydrogen bonding between water and the more basic diols. The inductive effect of the methyl group and longer chain alkyls is observed to increase basicity in ethylene glycol, propylene glycol, and 2,3-butanediol solutions. A correlation between the glass-forming ability of the diol solutions and the stronger hydrogen-bonding solutes (i.e., stronger bases) is developed, with 2,3-butanediol best promoting glass formation at the lowest concentrations.

13 C NMR spin–lattice relaxation times as a probeof local polymer dynamics in plasticized polyethers

¹³C NMR spin–lattice relaxation times T₁ are used to investigate the effect of low molecular weight diluents, including N,N-dimethylformamide, N-methylformamide, propylene carbonate, γ-butyrolactone, triglyme and tetraglyme, on the local polymer segmental motion in polyether–urethane networks. In all cases, an increase in the local mobility is deduced from the increasing T₁ measurements consistent with a decreasing glass transition temperature. The extent of plasticization, however, is dependent on the nature of the small molecules. Those molecules which can either form strong polymer-diluent interactions (for example through dipolar interactions) or are themselves rigid, give the least enhancement of polymer mobility and the greatest deviation from the Fox equation for T_g. In the presence of alkali metal salts, N,N-dimethylformamide and propylene carbonate are shown to have opposite effects on the local polymer motion, as seen from the T₁ measurements. In both cases, addition of the plasticizers increases the ¹³C T₁ relaxation times for the plasticizer. However, propylene carbonate decreases the polymer ¹³C T₁ whilst N,N-dimethylformamide results in the expected increase in polymer ¹³C T₁.

Dynamic mechanical thermal analysis studies of factors influencing relaxation in polyetherurethane based solid electrolytes

Dynamic mechanical thermal analysis (DMTA) has been used to study the effects of plasticizers on the mobility and homogeneity of a series of solid polymer electrolytes (SPEs). With reference to previously published results on similar systems containing LiClO₄ salts and tetraglyme as plasticizer, the effects of propylene carbonate (PC) on the glass transition temperature (T_g) of the SPE and on the distribution of relaxation times within the sample are discussed; at low plasticizer concentration PC has little effect on T_g as measured by DMTA in comparison with tetraglyme, and at higher plasticizer concentrations PC significantly broadens the mechanical relaxation behaviour indicating a greater degree of dynamical heterogeneity within the sample. A second low temperature relaxation is evident at lower PC contents indicating that some regions of this plasticized SPE are distinctly more mobile than others or perhaps, on this length scale, that some degree of phase separation is present. Activation energies for the mechanical relaxation were also determined as a function of PC concentration and are significantly greater than those determined from conductivity measurements.

Characterization of a proton conductor based on silicotungstic acid

It was found in this work that silicotungstic acid hydrate could be mixed with phosphoric acid (H₃PO₄, 85%) to make a viscous paste material with high conductivity (10⁻² S/cm at room temperature). The STA/H₃PO₄ paste samples were stable at 80°C in the atmosphere, and at 100°C under constant humidity over 10 days. The conductivity behavior of the paste samples has been investigated under various conditions, and it was found to be dependent on temperature, paste composition, and environment humidity.

A new class of proton-conducting ionic plastic crystals based on organic cations and dihydrogen phosphate

Choline dihydrogen phosphate ([N_1.1.1.2OH]DHP) and 1-butyl-3-methylimidazolium dihydrogen phosphate ([C₄mim]DHP) were synthesized as a new class of proton-conducting ionic plastic crystals. Both [N_1.1.1.2OH]DHP and [C₄mim]DHP showed solid–solid phase transition(s) and showed a final entropy of fusion lower than 20 J K⁻¹ mol⁻¹ which is consistent with Timmerman’s criterion for molecular plastic crystals. The ionic conductivity of [N_1.1.1.2OH]DHP was in the range of 10⁻⁶ S cm⁻¹–10⁻³ S cm⁻¹ in the plastic crystalline phase. On the other hand, the ionic conductivity of [C₄mim]DHP showed about 10⁻⁵ S cm⁻¹ in the plastic crystalline phase. [N_1.1.1.2OH]DHP showed one order of magnitude higher ionic conductivity than [C₄mim]DHP in the temperature range where the plastic phase is stable.

Investigation of proton dynamics and the proton transport pathway in choline dihydrogen phosphate using solid-state NMR

Choline dihydrogen phosphate has previously been shown to be a good ionic conductor as well as an excellent host for acid doping, leading to high proton conductivities required for e.g., electrochemical devices including proton membrane fuel cells and sensors. A combination of variable-temperature ¹H solid-state NMR and 2D NMR pulse sequences, including ³¹P and ¹³C CODEX and ¹H BaBa, show that the proton conduction mechanism primarily involves assisted transport via a restricted three-site motion of the phosphate unit around the P–O bond that is hydrogen bonded to the choline and exchange of protons between these anions. In other words, proton transport at ambient temperatures appears to occur most favorably along the crystallographic b axis, from phosphate dimer to dimer. At elevated temperatures exchange between the protons of the hydroxyl group on the choline cation and the hydrogen-bonded dihydrogen phosphate groups also contributes to the structural diffusion of the protons in this solid state conductor.

Proton transport in choline dihydrogen phosphate/H3PO4 mixtures

Mixtures of the plastic crystal material choline dihydrogen phosphate [Choline][DHP] and phosphoric acid, from 4.5 mol% to 18 mol% H₃PO₄, were investigated and shown to have significantly higher proton conductivity compared to the pure [Choline][DHP]. This was particularly evident from the electrochemical hydrogen reduction reaction and the proton NMR diffusion measurements, in addition to ionic conductivity measured from the impedance spectroscopy. The ionic conductivity was observed to increase by more than an order of magnitude in phase I (i.e. the highest temperature solid phase in [Choline][DHP]) reaching up to 10⁻² S cm⁻¹. The multinuclear NMR spectroscopy data suggest that, at least on the timescale of the NMR measurement, the H⁺ cations and [DHP] anions are equivalent in both phases. The pulsed field gradient NMR diffusion measurements of the 18 mol% acid sample indicate that all three ions are mobile, however the H⁺ diffusion coefficient is an order of magnitude higher than for the [Choline] cation or the [DHP] anion, and therefore conduction in these materials is dominated by proton conductivity. The thermal stability, as measured by TGA, is unaffected with increasing acid additions and remains high; i.e. no significant mass loss below 200 °C.

Identification of a by-product of nitric oxide synthase activity in human acute brain injury with in vivo proton magnetic resonance spectroscopy

BACKGROUND AND PURPOSE: Laboratory studies have been used to identify nitric oxide as a notable mediator in neuronal death after acute brain injury. To our knowledge, this has not previously been confirmed with in vivo study in humans. Our purpose was to seek in vivo evidence for the induction of nitric oxide synthase (NOS) in human acute brain injury by using proton MR spectroscopy.

METHODS: In vitro proton MR spectra were obtained in neural extracts from 30 human cadavers, and in vivo spectra were obtained in 20 patients with acute brain injury and in a similar number of control subjects.

RESULTS: We identified a unique peak at 3.15 ppm by using in vivo proton MR spectroscopy in eight of 20 patients with acute brain injury but not in 20 healthy volunteers (P < .002). On the basis of in vitro data, we have tentatively assigned this peak to citrulline, a NOS by-product.

CONCLUSION: To our knowledge, our findings suggest, for the first time, that excitotoxicity may occur in human acute brain injury. Confirmation with the acquisition of spectra in very early acute cerebral injury would provide a rationale for the use of neuroprotective agents in these conditions, as well as a new noninvasive method for quantification.

Proton transport properties in zwitterion blends with brønsted acids

We describe zwitterion, 3-(1-butyl-1H-imidazol-3-ium-3-yl)propane-1-sulfonate (Bimps), mixtures with 1,1,1-trifluoro-N-(trifluoromethylsulfonyl)methanesulfoneamide (HN(Tf)₂) as new proton transport electrolytes. We report proton transport mechanisms in the mixtures based on results from several methods including thermal analyses, the complex-impedance method, and the pulsed field gradient spin echo NMR (pfg-NMR) method. The glass transition temperature (Tg) of the mixtures decreased with increasing HN(Tf)₂ concentration up to 50 mol %. The Tg remained constant at −55 °C with further acid doping. The ionic conductivity of HN(Tf)₂ mixtures increased with the HN(Tf)₂ content up to 50 mol %. Beyond that ratio, the mixtures showed no increase in ionic conductivity (10⁻⁴ S cm⁻¹ at room temperature). This tendency agrees well with that of Tg. However, the self-diffusion coefficients obtained from the pfg-NMR method increased with HN(Tf)₂ content even above 50 mol % for all component ions. At HN(Tf)₂ 50 mol %, the proton diffusion of HN(Tf)₂ was the fastest in the mixture. These results suggest that Bimps cannot dissociate excess HN(Tf)₂, that is, the excess HN(Tf)₂ exists as molecular HN(Tf)₂ in the mixtures. The zwitterion, Bimps, forms a 1:1 complex with HN(Tf)₂ and the proton transport property in this mixture is superior to those of other mixing ratios. Furthermore, CH₃SO₃H and CF₃SO₃H were mixed with Bimps for comparison. Both systems showed a similar tendency, which differed from that of the HN(Tf)₂ system. The Tg decreased linearly with increasing acid content for every mixing ratio, while the ionic conductivity increased linearly. Proton transport properties in zwitterion/acid mixtures were strongly affected by the acid species added.