Structural and dynamical changes of the Streptococcus gordonii metalloregulatory ScaR protein induced by Mn2+ ion binding.

Divalent metal ions are essential micronutrients for many intercellular reactions. Maintaining their homeostasis is necessary for the survival of bacteria. In Streptococcus gordonii, one of the primary colonizers of the tooth surface, the cellular concentration of manganese ions (Mn2+) is regulated by the manganese-sensing transcriptional factor ScaR which controls the expression of proteins involved in manganese homeostasis. To resolve the molecular mechanism through which the binding of Mn2+ ions increases the binding affinity of ScaR to DNA, a variety of computational (QM and MD) and experimental (ITC, DSC, EMSA, EPR, and CD) methods were applied. The computational results showed that Mn2+ binding induces a conformational change in ScaR that primarily affects the position of the DNA binding domains and, consequently, the DNA binding affinity of the protein. In addition, experimental results revealed a 1:4 binding stoichiometry between ScaR dimer and Mn2+ ions, while the computational results showed that the binding of Mn2+ ions in the primary binding sites is sufficient to induce the observed conformational change of ScaR.

Improving the molecular spin qubit performance in zirconium MOF composites by mechanochemical dilution and fullerene encapsulation.

Enlarging the quantum coherence times and gaining control over quantum effects in real systems are fundamental for developing quantum technologies. Molecular electron spin qubits are particularly promising candidates for realizing quantum information processing due to their modularity and tunability. Still, there is a constant search for tools to increase their quantum coherence times. Here we present how the mechanochemical introduction of active spin qubits in the form of 10% diluted copper(ii)-porphyrins in the diamagnetic PCN-223 and MOF-525 zirconium-MOF polymorph pair can be achieved. Furthermore, the encapsulation of fullerene during the MOF synthesis directs the process exclusively toward the rare PCN-223 framework with a controllable amount of fullerene in the framework channels. In addition to the templating role, the incorporation of fullerene increases the electron spin-lattice and phase-memory relaxation times, T1 and Tm. Besides decreasing the amount of nuclear spin-bearing solvent guests in the non-activated qubit frameworks, the observed improved relaxation times can be rationalized by modulating the phonon density of states upon fullerene encapsulation.

A quantum many body model for the embedded electron spin decoherence in organic solids.

We present a generalized nuclear spin bath model for embedded electron spin decoherence in organic solids at low temperatures, which takes the crucial influence from hindered methyl group rotation tunneling into account. This new, quantum many body model, after resolved using the cluster correlation expansion method, predicts the decoherence profiles directly from the proton relative position and methyl group tunneling splitting inputs. Decoherence profiles from this model explain adequately the influence from both strongly and weakly hindered methyl groups to embedded electron spin decoherence: The former accelerates decoherence by increasing the nearest neighbor nuclear spin coupling, while the latter enhances coherence through a novel confinement like' mechanism, in which the very strong nuclear spin coupling from the tunneling splitting term suppresses those protons on the methyl rotors from participating in the bath dynamics. Both types of influences are successfully proven experimentally in representative organic polycrystalline matrices: methyl malonic acid for strongly hindered and acetamide for weakly hindered methyl groups, respectively.

Matrix material structure dependence of the embedded electron spin decoherence.

In this article, we present the novel application of the nuclear spin bath model and the cluster correlation expansion method on studying the matrix material structure via embedded electron spin decoherence. Profiles of embedded electron spin decoherence under the Carr-Purcell-Meiboom-Gill dynamical decoupling pulse series in a model system for organic solids (malonic acid) are calculated for different structures. Resulting decay profiles exhibit a strong correlation to the variations of an adjacent proton environment among them. In addition, the decoherence behavior of embedded spin in proton spin bath(s) of organic solids is found to be significantly different from bath models with other nuclei through the violation of the even-odd pulse parity, which characterizes the influence of large dipolar coupling between protons at the quantum level. Theoretical predictions of decoherence profiles in polycrystalline, the relative distribution of Hahn echo signal decay time scales among single crystal orientations, and the reduction in Hahn echo signal decay time scale by disorder are positively verified by experiments.

TROL-FNR interaction reveals alternative pathways of electron partitioning in photosynthesis.

In photosynthesis, final electron transfer from ferredoxin to NADP(+) is accomplished by the flavo enzyme ferredoxin:NADP(+) oxidoreductase (FNR). FNR is recruited to thylakoid membranes via integral membrane thylakoid rhodanase-like protein TROL. We address the fate of electrons downstream of photosystem I when TROL is absent. We have employed electron paramagnetic resonance (EPR) spectroscopy to study free radical formation and electron partitioning in TROL-depleted chloroplasts. DMPO was used to detect superoxide anion (O2(.-)) formation, while the generation of other free radicals was monitored by Tiron. Chloroplasts from trol plants pre-acclimated to different light conditions consistently exhibited diminished O2(.-) accumulation. Generation of other radical forms was elevated in trol chloroplasts in all tested conditions, except for the plants pre-acclimated to high-light. Remarkably, dark- and growth light-acclimated trol chloroplasts were resilient to O2(.-) generation induced by methyl-viologen. We propose that the dynamic binding and release of FNR from TROL can control the flow of photosynthetic electrons prior to activation of the pseudo-cyclic electron transfer pathway.

The effect of thermal treatment of radiation-induced EPR signals of different polymorphic forms of trehalose.

Electron paramagnetic resonance (EPR) signals induced by γ-radiation in different polymorphic forms of trehalose were studied with dosimetry applications in view. Dose response of trehalose in terms of the concentration of induced paramagnetic centers was studied in the dose range from 0.5 to 50 kGy. The dependences of the dose responses of anhydrous ß-crystalline trehalose (TRE(ß)) and glassy trehalose (TRE(g)) on dose are linear up to 15 kGy, whereas the linearity of the dependence for trehalose dihydrate (TRE(h)) is limited to about 10 kGy. At doses above 15 kGy, the dependences get saturated for all three forms. The relative radiation sensitivities pointed to the following order of decreasing concentrations of radiation-induced paramagnetic centers in the forms: TRE(g)>TRE(ß)>TRE(h). The results showed that at all three trehalose polymorphic forms are suitable for dosimetry, especially for retrospective dose measurements. Also, thermal stability and decay kinetics of the EPR signals of the different forms of trehalose were studied in isothermal annealing experiments. The kinetic parameters, which had been derived by fitting the Arrhenius function to the measured decay rate constants, indicated that the fading of the EPR signals varied from one polymorphic form of trehalose to another. This emphasizes the impact of the molecular packing in the vicinity of the radiation-induced paramagnetic centers on their stability.

Effect of glassy modes on electron spin-lattice relaxation in solid ethanol.

Electron spin-lattice relaxation (SLR) of TEMPO radical was measured in the crystalline and glassy states of deuterated ethanol in the temperature range 5-80K using X-band electron paramagnetic resonance (EPR). The measured SLR rates are higher in the glassy than in crystalline state and the excess SLR rate in glassy state is much lower than in ethanol. This result suggests that extra modes in glassy state, i.e. glassy modes, produce the excess SLR rate via the electron-nuclear dipolar (END) interaction between the electron spin of radical and the matrix protons or deuterons. Using the soft-potential model and assuming the END interaction between the electron spin and the matrix protons, the contributions to SLR rate of various mechanisms of glassy modes were theoretically analyzed. The evaluations of SLR rates in glassy ethanol indicate two main mechanisms of glassy modes: thermally activated relaxation of double-well systems and phonon-induced relaxation of quasi-harmonic local modes. The SLR rates induced by these mechanisms correlate well with the experimental data.

A multi-frequency EPR spectroscopy approach in the detection of boson peak excitations.

The influence of boson peak (BP) excitations on low-temperature spin-lattice relaxation rate of a paramagnetic center embedded in a glassy matrix is investigated in the context of multi-frequency electron paramagnetic resonance (EPR) detection. In the theoretical analysis, the transition rate of spin one-half in the presence of a phonon field is calculated within the approximation of Fermi's golden rule. Several phonon densities of states are compared, among which one originating from a model of quasi-localized vibrations has been introduced into electron spin relaxation formalism for the first time. The respective frequency dependencies of spin-lattice relaxation rates are predicted which should lead to observable effects of BP modes if a multi-frequency study at very low temperatures is performed.

Fast motion in molecular solids at low temperatures: evidence from a pulsed electron paramagnetic resonance study of nitroxyl radical relaxation.

We have investigated the electron phase-memory relaxation time of the nitroxyl radical 2,2,6,6-tetramethylpiperidine-1-oxyl at temperatures between 5 and 80 K in crystalline and glassy states of ethanol using pulsed X-band electron paramagnetic resonance spectroscopy. The results indicate that the transition from the slow to fast motion regimes of the paramagnetic center occurs upon further cooling of the sample below ∼20 K. We provide experimental evidence that this phenomenon cannot be ascribed to the impact of hyperfine interactions with methyl protons in the system, but it can be instead a signature of the coupling of the electron spin with the boson peak excitations of the lattice.

Antioxidant properties of ganglioside micelles.

Antioxidant activity of gangliosides GM1 and GT1b in the Fenton type of reaction was investigated by EPR spectroscopy using DMPO as a spin trap. Hydroxyl radical spin adduct signal intensity was significantly reduced in the presence of gangliosides at their micellar concentrations. Mean micellar hydrodynamic diameter was not changed, whereas significant changes in negative Zeta potential values were observed as evidenced by Zetasizer Nano ZS. This study showed that the primary mode of ganglioside action was not due to direct scavenging of OH., but rather to the inhibition of hydroxyl radical formation. This phenomenon is related to the ability of ganglioside micelles to bind oppositely charged ferrous ions, thus reducing their concentration and consequently inhibiting OH. formation.

Interaction of alcohols with serum LDL An infrared study.

The interaction of low molecular weight alcohols with low density lipoprotein (LDL) has been studied using amide I band-fitting, thermal profiling and two-dimensional infrared correlation spectroscopy (2D-IR). At 0.3 M alcohol, no changes in secondary structure are observed. In the presence of 1 M alcohol, ethanol and propanol decreases protein denaturation temperature and produces changes in the amide I thermal profiles of protein components and in the lipid bands. The 2D-IR synchronous map corresponding to protein or lipid component at 20-37 degrees C suggests differences in the presence of propanol. The asynchronous map corresponding to the lipid component indicates changes in bandwidth, compatible with a more fluid environment. In the 37-80 degrees C temperature range the thermal profile is different in the presence of propanol, both for the lipid and protein components. The results presented show that when alcohols affect the protein component, the lipid spectrum also varies pointing to an effect on the lipid-protein interaction.

Lipid-protein interactions in human plasma LDL evidenced by magnetic resonance.

Low density lipoprotein (LDL) particles exhibit extremely complex three-dimensional structural organization which is still not understood at the molecular level. The aim of this study was to provide the experimental evidence of a direct non-covalent interaction of the protein part with the lipid matrix. The approach was based on the combination of (1)H NMR (600 MHz) spectroscopy with thiol-specific spin labeling of the protein (apoB). It is shown that the spectral peaks assigned to the methyl head groups of phosphatidylcholine and sphingomyelin in the (1)H spectra of LDL exhibit line broadening when otherwise free thiol groups of apoB are covalently modified by methanethiosulfonate spin label. The effect is similar in the presence of water soluble paramagnetic compound. These results indicate that fragments of apoB, which are part of the receptor binding region, are directly in contact with the solvated phospholipid head groups of the lipid matrix.

The effect of heparin on structural and functional properties of low density lipoproteins.

Heparin binding to human low density lipoproteins (LDL) and the effect of heparin on the ability of LDL to bind to the LDL receptor has been investigated. Emphasis has been made on the physiological conditions of temperature, pH and the ionic strength. Intrinsic fluorescence spectroscopy of LDL has been applied to follow heparin binding. Fluorescence anisotropy has been measured to describe the changes in apoB and dansyl-heparin dynamics upon binding. Eu3+-labeled LDL binding to the intact LDL receptor has been monitored by time-resolved fluorescence spectroscopy technique. We have found that heparin binds to LDL under the physiological conditions, probably by Van der Waals interactions and hydrogen bonding. Temperature seems to be the most important factor influencing the interaction. Furthermore, the presence of heparin inhibits LDL binding to the intact LDL receptor that might have consequences on the cholesterol metabolism in vivo.

Trisialoganglioside GT1b prevents increase in sperm membrane molecular ordering induced by in vitro lipid peroxidation.

The effect of various types of gangliosides, the sialic acid-containing glycosphingolipids, on human sperm membrane during lipid peroxidation induced by Fe(2+)/ascorbate ions was investigated. The monosialoganglioside (GM1), disialogangliosides (GD1a and GD1b), and trisialoganglioside (GT1b) were examined at a concentration of 100 microM, which was above their respective critical micellar concentrations. Lipid peroxidation was determined by quantification of malondialdehyde (MDA) concentration. The molecular orientational order in the membrane was assessed by fluorescence spectroscopy and electron paramagnetic resonance spectroscopy. Both approaches revealed a significant increase in membrane rigidity following oxidation, which correlated with an increase in the MDA level. The preincubation of spermatozoa with GM1 and GD1a did not have any effect on induced lipid peroxidation. In the presence of GD1b and GT1b, a reduced formation of MDA and a decrease in membrane rigidity was detected. The inhibitory effect of GT1b micelles toward membrane oxidation damage was found to be greater than that of GD1b. In conclusion, a direct relationship between the reduced content of the accumulated MDA and the longer preservation of the native-like membrane molecular ordering during sperm oxidation in the presence of GT1b suggests its protective effect. This phenomenon could be due to the specific GT1b conformation and its negative surface potential.

Effect of caffeine on oxidation susceptibility of human plasma low density lipoproteins.

The effect of caffeine on oxidation susceptibility of low density lipoproteins (LDL) has been studied. LDL oxidation was induced by copper ions and an azo initiator. The conjugated dienes formation was followed spectrophotometrically and indicated the LDL oxidation status. Changes in LDL protein moiety during the lag phase, studied only in the experiments of copper induced oxidation, were followed using the intrinsic fluorescence spectroscopy. The decay of LDL fluorescence signal during initial stages of oxidation was slower in the presence of caffeine. Supported by the fluorescence quenching and polarization measurements, these results may indicate the protective role of caffeine against LDL oxidation in vitro. The results also indicate that the production of conjugated dienes in the propagation and decomposition phase of LDL oxidation is lower in the presence of caffeine, regardless of the initiation mechanism. These findings may have implications for the effect of caffeine on LDL in vivo.

Absorption and fluorescence spectra of ring-substituted indole-3-acetic acids.

The absorption and fluorescence spectra of indole-3-acetic acid (1), a plant growth regulator (auxin) and experimental cancer therapeutic, 29 ring-substituted derivatives and the 7-aza analogue (1H-pyrrolo[2,3b]pyridine-3-acetic acid) are compared. Two to four absorbance maxima in the 260-310-nm range are interpreted as overlapping vibronic lines of the 1La<--1A and 1Lb<--1A transitions. Two further maxima in the 200-230-nm region are assigned to the 1Ba<--1A and 1Bb<--1A transitions. 4- and 7-Fluoroindole-3-acetic acid exhibit blue shifts with respect to 1, most other derivatives show red shifts. All indole-3-acetic acids studied, with the exception of chloro-, bromo- and 4- or 7-fluoro-derivatives, fluoresce at 345-370 nm when excited at 275-280 nm. 7-Azaindole-3-acetic acid emits at 411 nm. The fluorescence quantum yield of 6-fluoroindole-3-acetic acid significantly exceeds that of 1 (0.3); the other derivatives have lower quantum yields. The plant-growth promoting activity of the ring-substituted indole-3-acetic acids studied correlates with the position of the 1Bb<--1A transition band.

Analysis of beta-carotene absorbance for studying structural properties of human plasma low-density lipoproteins.

A novel spectrophotometric assay for monitoring structural rearrangements of native low-density lipoproteins (LDL) is proposed. The approach is based on the analysis of the visible light absorbance maximum of lipoproteins at approximately 461 nm assigned to beta-carotene situated in the hydrophobic parts of LDL. It offers a direct method to study the surface-interior coupling of the lipoprotein particle under physiological conditions. The detected signal is intrinsic to LDL and responsible for the most of the beta-carotene signal from the whole plasma. The negligible interference of beta-carotene absorbance due to the high-density lipoproteins is experimentally verified. Since beta-carotene absorbance belongs to the visible spectral region, no spectral overlapping/artifacts in plasma are expected. The signal sensitivity has been studied through conformational changes of LDL induced by ionic strength, by temperature, and by ligand binding. The results of caffeine binding to LDL indicate that there could be only one dominant type of binding site for caffeine on LDL particles. It can be concluded that visible spectrum characteristics of beta-carotene molecules offer advantages in LDL ligand binding studies which can possibly be extended to monitor the interactions of LDL directly in plasma.

Effect of gangliosides on the copper-induced oxidation of human low-density lipoproteins.

The role of gangliosides in the copper-induced oxidative modification of human low-density lipoprotein (LDL) was studied focusing on the early stage of LDL oxidation in which the concentration of conjugated dienes increases only weakly. The changes in the protein and lipid component were followed using fluorescence spectroscopy. The results indicate that binding of gangliosides to LDL causes slower destruction of tryptophan fluorescence and suppresses cross-linking between the reactive groups of the protein and the products of lipid peroxidation. The protective role of gangliosides could be assigned to their interference with the lipid-protein interaction in the LDL particle, which might be important for the maintenance of the native plasma antioxidant status in vivo.

The study of structural accessibility of free thiol groups in human low-density lipoproteins.

The experimental evidence for the apolipoprotein B100 (apoB) domain structuring in low-density lipoprotein (LDL) was investigated focusing on the accessibility of free thiol groups. Three different spectroscopic methods were combined with the biochemical perturbations of LDL particle. The spectrophotometric method was adapted for LDL and the exposure of free thiols was analyzed in the native LDL and LDL exposed to sequential denaturation. The results indicate that 24-h denaturation does not expose all free thiols in LDL. Using thiol-specific spin labeling and electron paramagnetic resonance spectroscopy (EPR), different populations of labeled thiols were resolved. The comparison of the EPR spectra of native LDL and LDL with selectively blocked thiol groups revealed significant difference in the respective hyperfine splittings. The phenomenon can arise due to different polarity and/or mobility of the nitroxides in the microenvironments of spin label binding sites of these two LDL samples. The results indicate that nine thiol groups in apoB are distributed in different domains of LDL: two are more exposed, two are buried deeply in the lipid matrix of the particle and the rest are located in hydrophobic parts of this extremely complex protein-lipid assembly. These observations provide experimental support for the emerging theoretical models of apoB.