Performance of wave function and density functional methods for water hydrogen bond spin–spin coupling constants

Spin–spin coupling constants in water monomer and dimer have been calculated using several wave function and density functional-based methods. CCSD, MCSCF, and SOPPA wave functions methods yield similar results, specially when an additive approach is used with the MCSCF. Several functionals have been used to analyze their performance with the Jacob’s ladder and a set of functionals with different HF exchange were tested. Functionals with large HF exchange appropriately predict ¹ J _{O H} , ² J _{H H} and ^2h J _{O O} couplings, while ^1h J _{O H} is better calculated with functionals that include a reduced fraction of HF exchange. Accurate functionals for ¹ J _{O H} and ² J _{H H} have been tested in a tetramer water model. The hydrogen bond effects on these intramolecular couplings are additive when they are calculated by SOPPA(CCSD) wave function and DFT methods.

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Graphical Abstract Evaluation of the additive effect of the hydrogen bond on spin-spin coupling constants of water using WF and DFT methods.

     

GroEL-induced topological dislocation of a substrate protein β-sheet core: a solution EPR spin–spin distance study

The Hsp60-type chaperonin GroEL assists in the folding of the enzyme human carbonic anhydrase II (HCA II) and protects it from aggregation. This study was aimed to monitor conformational rearrangement of the substrate protein during the initial GroEL capture (in the absence of ATP) of the thermally unfolded HCA II molten-globule. Single- and double-cysteine mutants were specifically spin-labeled at a topological breakpoint in the β-sheet rich core of HCA II, where the dominating antiparallel β-sheet is broken and β-strands 6 and 7 are parallel. Electron paramagnetic resonance (EPR) was used to monitor the GroEL-induced structural changes in this region of HCA II during thermal denaturation. Both qualitative analysis of the EPR spectra and refined inter-residue distance calculations based on magnetic dipolar interaction show that the spin-labeled positions F147C and K213C are in proximity in the native state of HCA II at 20 °C (as close as ～8 Å), and that this local structure is virtually intact in the thermally induced molten-globule state that binds to GroEL. In the absence of GroEL, the molten globule of HCA II irreversibly aggregates. In contrast, a substantial increase in spin–spin distance (up to >20 Å) was observed within minutes, upon interaction with GroEL (at 50 and 60 °C), which demonstrates a GroEL-induced conformational change in HCA II. The GroEL binding-induced disentanglement of the substrate protein core at the topological break-point is likely a key event for rearrangement of this potent aggregation initiation site, and hence, this conformational change averts HCA II misfolding.     

Electron spin resonance and spin trapping technique provide direct evidence that edaravone prevents acute ischemia–reperfusion injury of the liver by limiting free radical-mediated tissue damage

A novel free radical scavenger, 3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone), is used for the treatment of acute ischemic stroke and is protective in several animal models of organ injury. We tested whether edaravone is protective against acute liver warm ischemia/reperfusion injury in the rat by acting as a radical scavenger. When edaravone was administered prior to ischemia and at the time of initiation of the reperfusion, liver injury was markedly reduced. Production of oxidants in the liver in this model was assessed in vivo by spin-trapping/electron spin resonance (ESR) spectroscopy. Ischemia/reperfusion caused an increase in free radical adducts rapidly, an effect markedly blocked by edaravone. Furthermore, edaravone treatment blunted ischemia/reperfusion-induced elevation in pro-inflammatory cytokines, infiltration of leukocytes and lipid peroxidation in the liver. These results demonstrate that edaravone is an effective blocker of free radicals in vivo in the liver after ischemia/reperfusion, leading to prevention of organ injury by limiting the deleterious effects of free radicals.     

Accelerating proton spin diffusion in perdeuterated proteins at 100 kHz MAS

Fast magic-angle spinning (>60 kHz) has many advantages but makes spin-diffusion-type proton–proton long-range polarization transfer inefficient and highly dependent on chemical-shift offset. Using 100%-HN-[²H,¹³C,¹⁵N]-ubiquitin as a model substance, we quantify the influence of the chemical-shift difference on the spin diffusion between proton spins and compare two experiments which lead to an improved chemical-shift compensation of the transfer: rotating-frame spin diffusion and a new experiment, reverse amplitude-modulated MIRROR. Both approaches enable broadband spin diffusion, but the application of the first variant is limited due to fast spin relaxation in the rotating frame. The reverse MIRROR experiment, in contrast, is a promising candidate for the determination of structurally relevant distance restraints. The applied tailored rf-irradiation schemes allow full control over the range of recoupled chemical shifts and efficiently drive spin diffusion. Here, the relevant relaxation time is the larger longitudinal relaxation time, which leads to a higher signal-to-noise ratio in the spectra.     

Marqueurs Á spin construits directement sur le squelette carboné de sucres

Treatment of aldehydo-sugars with 2,3-bis(hydroxyamino)-2,3-dimethylbutane gave the corresponding 1,3-dihydroxyimidazolidines. Oxidation of these compounds gave, successively, unstable, free radicals having a 3-hydroxyimidazolidine 1-oxyl structure, then stable, free radicals having a 2-glycosyl-2-imidazoline 3-oxide 1-oxyl structure, which were reduced to stable 2-glycosyl-2-imidazoline 1-oxyl compounds. The orientation of the two last-named series of compounds around the σ bond between carbohydrate and heterocyclic residues that bears the radical centre is indicated by the value of the hyperfine coupling aH of the carbohydrate proton nearest to the nitrogenous heterocyclic residue. The study of numerous compounds of both series shows that the value of the hyperfine coupling depends greatly on the structure of the carbohydrate residue. A general explanation based on the preponderance of eclipsed forms and on the primary influence of steric hindrance due to the substituents of the carbohydrate residue is proposed. This study also gives some indirect information on the conformation of diamagnetic compounds that show little sterical difference, and about which ¹H-n.m.r. spectroscopy does not provide useful information.     

Photoreduction of the fluorescent dye 2′-7′-dichlorofluorescein: a spin trapping and direct electron spin resonance study with implications for oxidative stress measurements

The photoreduction of 2′-7′-dichlorofluorescein (DCF) was investigated in buffer solution using direct electron spin resonance (ESR) and the ESR spin-trapping technique. Anaerobic studies of the reaction of DCF in the presence of reducing agents demonstrated that during visible irradiation (λ > 300 nm) 2′-7′-dichlorofluorescein undergoes one-electron reduction to produce a semiquinone-type free radical as demonstrated by direct ESR. Spin-trapping studies of incubations containing DCF, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and either reduced glutathione (GSH) or reduced NADH demonstrate, under irradiation with visible light, the production of the superoxide dismutase-sensitive DMPO/^·OOH adduct. In the absence of DMPO, measurements with a Clark-type oxygen electrode show that molecular oxygen is consumed in a light-dependent process. The semiquinone radical of DCF, when formed in an aerobic system, is immediately oxidized by oxygen, which regenerates the dye and forms superoxide.     

Does 3,5,dibromo-4-nitrosobenzene sulphonate spin trap superoxide radicals?

Pulse radiolysis studies show that the spin trap 3,5,dibromo-4-nitrosobenzene sulphonate (I) reacts rapidly with O2.- but the product formed is very unstable. No radicals were detected in ESR studies of solutions of I after reaction with O2.- formed by gamma-radiolysis. Evidence is presented that the stable radical observed by some, but not all workers, following exposure of I to the O2.(-)-generating xanthine/xanthine oxidase system, is produced by a peroxidatic oxidation using hydrogen peroxide formed by O2-. dismutation and that formation of this radical depends on the presence of peroxidase activity in the xanthine oxidase sample employed.     

Electron spin resonance studies on the dynamics of phosphatidylcholine — sulfatide model membranes

The effects of sulfatide on the fluidity and surface dynamics of bilayered and micellar model membranes of egg phosphatidylcholine containing sulfatide were studied by electron spin resonance (ESR). 5-Nitroxystearic acid and 15-nitroxystearic acid were employed as spin-label probes for the region close to the surface and that close to the hydrophobic core of lipid structures. In the vesicular structures, the signals generated by 5-nitroxystearic acid showed that the presence of sulfatide reduced the mobility of the hydrocarbon chains around the probe. The effect increased with increasing glycolipid concentration. The decrease in membrane fluidity was also monitored with the 15-nitroxystearic acid probe, although to a lesser extent. We think that sulfatide causes strong side-to-side head-group interactions on the bilayer surface, causing the lipid chains to assemble in a more rigid fashion, though this effect may be balanced in part by the disordered mechanical coupling of glycolipid acyl chains in theapposite faces of the hydrophobic core of the bilayer. Reduction of this mechanical coupling between apposite lipids when there was transition from a bilayered to a micellar structure resulted in a further increase in the order of the system.     

Synthesis and biological evaluation of novel spin labeled 18β-glycyrrhetinic acid derivatives

Eighteen novel spin-labeled 18β-glycyrrhetinic acid (GA) derivatives were designed, synthesized, and evaluated for cytotoxicity against four human tumor cell lines (A-549, DU-145, KB and KBvin). Most of the derivatives showed more significant cytotoxicity than that of the parent compound GA. The best compound, 6j, with a tryptophan amino moiety and piperidine nitroxyl radical showed GI₅₀ values of 13.7–15.0 μM, and was fivefold more potent than GA. In a mechanism of action study, compound 7a was confirmed as a 20S proteasome inhibitor in both in vitro and cell-based assays. These findings support further optimization efforts based on 18β-GA as a lead compound to develop potential anticancer drug candidates.     

Local spin dynamics in magnetic molecular chains studied by NMR and μSR

We present NMR and μ⁺SR study of spin dynamics in one-dimensional and quasi-one-dimensional molecular magnets of recent synthesis. In particular, we focus on the so called Gd(hfac)₃NIT-R and CoPhOMe magnetic chains families. For Gd-R helimagnets we show some differences between “weakly frustrated systems” and “fully frustrated systems”. The different behaviour is due to the different radical inserted in the chains (R = Me, Ph for “weakly frustrated systems” and R = iPr, Et for “fully frustrated systems”). The existence of different phase transitions, particularly to 3D long-range magnetic order in Gd-Ph and to chiral order in Gd-iPr, is remarked together with a comparison between results obtained from macroscopic and microscopic investigating techniques. As regards CoPhOMe slowly relaxing chain, the ¹H NMR measurements confirm the freezing of the spins at low temperature, which prevents the 3D long-range order, and display the presence of two relaxation mechanisms related to distinct contributions to the local spin relaxation.     

Is the Fab-fragment from monoclonal rheumatoid IgM flexible? A spin label dynamics study

A comparative study of the Fab- and Fab-RF-fragments in the molecules of monoclonal IgM and IgM-RF, respectively, was performed by the spin label method. The spin label, 2,2,6,6-tetramethyl-4-dichloro-triazinylaminopiperidine-1-oxyl, was introduced into the peptide part of the protein. On the basis of the data on the temperature-viscosity dependences of the EPR spectral parameters of the resultant spin-labeled proteins, the rotational correlation time tau of the spin carrier was determined. It turned out that the reduced to normal conditions tau values for the molecules of the Fab- and Fab-RF-fragments were 21+/-2 and 11+/-1 ns, respectively. Analysis of the resultant data provides sufficient grounds for assuming that such a sharp decrease in the tau value for the molecule of the Fab-RF fragment is due to local flexibility of its globular structure, which, in turn, can determine the specific features of the IgM-RF functioning as an autoantibody.     

Characteristics and use as spin trapping agent of a β-phosphorylated nitroso compound,DEPNP

2-(Diethylphosphonate)-nitrosopropane (DEPNP), prepared by oxidation of the corresponding aminophosphonate, was found to essentially exist as monomer in both water and organic solvents. The mechanisms of its degradation under 80°C heating or visible light exposure were studied by EPR spectroscopy: its decomposition gave rise to paramagnetic by-products, which have been identified as DEPNP / ·C(CH₃)₂[P(O)(OC₂H₅)₂] and DEPNP / ·P(O)(OC₂H₅)₂ spin adducts. Despite this drawback, DEPNP was successfully used as spin trapping agents to scavenge various carbon — and phosphorus-centred free radicals both in aqueous and organic media, giving rise to intense EPR spectra characteristic of the species trapped.     

Does propofol alter membrane fluidity at clinically relevant concentrations? An ESR spin label study

General anesthetics have been shown to perturb the membrane properties of excitable tissues. Due to their lipid solubility, anesthetics dissolve in every membrane, penetrate into organelles and interact with numerous cellular structures in multiple ways. Several studies indicate that anesthetics alter membrane fluidity and decrease the phase-transition temperature. However, the required concentrations to induce such effects on the properties of membrane lipids are by far higher than clinically relevant concentrations. In the present study, the fluidizing effect of the anesthetic agent propofol (2,6-diisopropyl phenol: PPF), a general anesthetic extensively used in clinical practice, has been investigated on liposome dimyristoyl-L-alpha phosphatidylcholine (DMPC) and cell (erythrocyte, Neuro-2a) membranes using electron spin resonance spectroscopy (ESR) of nitroxide labeled fatty acid probes (5-, 16-doxyl stearic acid). A clear effect of PPF at concentrations higher than the clinically relevant ones was quantified both in liposome and cell membranes, while no evident fluidity effect was measured at the clinical PPF doses. However, absorption spectroscopy of merocyanine 540 (MC540) clearly indicates a PPF fluidizing capacity in liposome membrane even at these clinical concentrations. PPF may locally influence the structure and dynamics of membrane domains, through the formation of small-scale lipid domains, which would explain the lack of ESR information at low PPF concentrations.     

Model membrane partition ESR study in the presence ofα-tocopherol by a new spin probe

The effect of-tocopherol (T) on partitioning and fluidity changes occurring in phospholipid liposomes have been investigated by monitoring the X-band ESR spectrum of the high resolution amphiphilic spin probe perdeutero-di-t-butyl nitroxide (PDDTBN), which partitions in the lipid and water phase of liposomes, showing all the three resonances from each phase well resolved.     

Zero field resonance and spin alignment of the triplet state of chloroplasts at 2°K

Microwave induced transitions in zero magnetic field have been observed in the photoinduced triplet of chloroplasts treated with dithionite by monitoring changes in the intensity of the 735 nm fluorescence band at 2°K. Similar results were obtained with chloroplasts treated with hydroxylamine plus 3-(3,4-dichlorophenyl)-1,1-dimethylurea and preillumination. The zero field parameters are $\text{D = 0.02794 ± 0.00007}\text{cm}{}^{\mathrm{?1}}$, $\text{E = 0.00382 ± 0.00007}\text{cm}{}^{\mathrm{?1}}$, i.e. equal to those of monomeric chlorophyll $\text{a}$ to within the experimental error. The photoinduced triplet appears to be linked to Photosystem II. This indicates that the low temperature 735 nm fluorescence band of chloroplasts is at least partly due to Photosystem II.     

Heteronuclear relaxation in time-dependent spin systems: 15N-T1ρ dispersion during adiabatic fast passage

A novel NMR experiment comprising adiabatic fast passage techniques for the measurement of heteronuclear self-relaxation rates in fully 15N-enriched proteins is described. Heteronuclear self-relaxation is monitored by performing adiabatic fast passage (AFP) experiments at variable adiabaticity (e.g., variation of RF spin-lock field intensity). The experiment encompasses gradient- selection and sensitivity-enhancement. It is shown that transverse relaxation rates derived with this method are in good agreement with the ones measured by the classical Carr–Purcell–Meiboom–Gill (CPMG) sequences. An application of this method to the study of the carboxyl-terminal LIM domain of quail cysteine and glycine-rich protein qCRP2(LIM2) is presented.     

Pulsed electron spin resonance resolves the coordination site of Cu²(+) ions in α1-glycine receptor

Herein, we identify the coordination environment of Cu²⁺ in the human α1-glycine receptor (GlyR). GlyRs are members of the pentameric ligand-gated ion channel superfamily (pLGIC) that mediate fast signaling at synapses. Metal ions like Zn²⁺ and Cu²⁺ significantly modulate the activity of pLGICs, and metal ion coordination is essential for proper physiological postsynaptic inhibition by GlyR in vivo. Zn²⁺ can either potentiate or inhibit GlyR activity depending on its concentration, while Cu²⁺ is inhibitory. To better understand the molecular basis of the inhibitory effect we have used electron spin resonance to directly examine Cu²⁺ coordination and stoichiometry. We show that Cu²⁺ has one binding site per α1 subunit, and that five Cu²⁺ can be coordinated per GlyR. Cu²⁺ binds to E192 and H215 in each subunit of GlyR with a 40 μM apparent dissociation constant, consistent with earlier functional measurements. However, the coordination site does not include several residues of the agonist/antagonist binding site that were previously suggested to have roles in Cu²⁺ coordination by functional measurements. Intriguingly, the E192/H215 site has been proposed as the potentiating Zn²⁺ site. The opposing modulatory actions of these cations at a shared binding site highlight the sensitive allosteric nature of GlyR.     

Distribution of effective field in the ising spin glass of the ±J model atT=0

The integral equation for the distribution function of effective field of the ±J random Ising model in the pair (Bethe) approximation is investigated. Its exact solutions atH (magnetic field)=0,T (temperature)=0 and forz (coordination number)=3 expressed as superpositions of 2N+1 (more than 3), delta functions are considered. Then the integral equation is reduced to a system of algebraic equations ofz ?1th degree withN+1 unknowns. The system of the equations is solved by the Gröbner basis method withN=1, 2, 3, 4. The number of physically acceptable solutions for a givenN is ω(N)+1 where ω(N) is the number of divisors ofN. The ground-state energy and entropy for these are calculated. They are very close in value (entropies are positive), and it is suggested that a number of physically acceptable solutions correspond to local stationary spin-glass states, as discussed in the literatures.