Investigations on unconventional hydrogen bonds in RNA binding proteins: The role of CH⋯OC interactions

We have investigated the roles played by CH⋯OC interactions in RNA binding proteins. There was an average of 78 CH⋯OC interactions per protein and also there was an average of one significant CH⋯OC interaction for every 6 residues in the 59 RNA binding proteins studied. Main chain–Main chain (MM) CH⋯OC interactions are the predominant type of interactions in RNA binding proteins. The donor atom contribution to CH⋯OC interactions was mainly from aliphatic residues. The acceptor atom contribution for MM CH⋯OC interactions was mainly from Val, Phe, Leu, Ile, Arg and Ala. The secondary structure preference analysis of CH⋯OC interacting residues showed that, Arg, Gln, Glu and Tyr preferred to be in helix, while Ala, Asp, Cys, Gly, Ile, Leu, Lys, Met, Phe, Trp and Val preferred to be in strand conformation. Most of the CH⋯OC interacting polar amino acid residues were solvent exposed while, majority of the CH⋯OC interacting non polar residues were excluded from the solvent. Long and medium-range CH⋯OC interactions are the predominant type of interactions in RNA binding proteins. More than 50% of CH⋯OC interacting residues had a higher conservation score. Significant percentage of CH⋯OC interacting residues had one or more stabilization centers. Sixty-six percent of the theoretically predicted stabilizing residues were also involved in CH⋯OC interactions and hence these residues may also contribute additional stability to RNA binding proteins.     

Water-soluble N-carboxymethylchitosan derivatives: Preparation,characteristics and its application

In this work, only N-substituted chitosan derivatives (water-soluble N-carboxymethylchitosan derivatives: N-CMC) with different degrees of substitution were obtained by reaction of a fully deacetylated chitosan (derived from deacetylation of chitosan using decrystallized method) with monochloroacetic acid at pH 8 and temperature of 90 °C. The structure of N-carboxymethylchitosan and chitosan was characterized by IR, ¹H, ¹³C and ¹H–¹³C NMR-HSQC spectra. In the IR spectrum of the N-carboxymethylchitosan, the appearance of peak at 1742 cm^?1 was assigned for CO group of NHCH₂COOH of substituted chitosan. In the ¹H NMR spectra, the peaks at about 3.81÷4.06 ppm, assigned for CH₂ groups of NHCH₂ and N(CH₂)₂, were the major feature, while in the ¹H–¹³C NMR-HSQC spectra, signals of CH₂ confirmed the presence of these two different substituted CH₂ groups. The degree of substitution (DS) of N-monosubstitution (DS_N-mono) decreased from 0.47 to 0.03 meanwhile that of N,N-disubstitution (DS_N,N-di) increased from 0.52 to 0.96 since the mass ratio of chitosan/monochloroacetic acid changing from 1/1 to 1/4. The N-carboxymethylchitosan derivatives have been used for adsorption Cu(II) ion from aqueous solution. The results shown that the optimum conditions for adsorption Cu(II) ion in nitrate solution were pH 6.5, temperature of 30 °C, for 60–90 min and the substituted chitosan derivative having DS_N-mono of 0.16 and DS_N,N-di of 0.81 had maximum adsorption capacity of 192 mg Cu(II) per gram of N-CMC.     

Intramolecular C(sp3)H amination of arylsulfonyl azides with engineered and artificial myoglobin-based catalysts

The direct conversion of aliphatic CH bonds into CN bonds provides an attractive approach to the introduction of nitrogen-containing functionalities in organic molecules. Following the recent discovery that cytochrome P450 enzymes can catalyze the cyclization of arylsulfonyl azide compounds via an intramolecular C(sp³)H amination reaction, we have explored here the CH amination reactivity of other hemoproteins. Various heme-containing proteins, and in particular myoglobin and horseradish peroxidase, were found to be capable of catalyzing this transformation. Based on this finding, a series of engineered and artificial myoglobin variants containing active site mutations and non-native Mn- and Co-protoporphyrin IX cofactors, respectively, were prepared to investigate the effect of these structural changes on the catalytic activity and selectivity of these catalysts. Our studies showed that metallo-substituted myoglobins constitute viable CH amination catalysts, revealing a distinctive reactivity trend as compared to synthetic metalloporphyrin counterparts. On the other hand, amino acid substitutions at the level of the heme pocket were found to be beneficial toward improving the stereo- and enantioselectivity of these Mb-catalyzed reactions. Mechanistic studies involving kinetic isotope effect experiments indicate that CH bond cleavage is implicated in the rate-limiting step of myoglobin-catalyzed amination of arylsulfonyl azides. Altogether, these studies indicate that myoglobin constitutes a promising scaffold for the design and development of CH amination catalysts.     

Precursor De13.1 from Conus delessertii defines the novel G gene superfamily

Peptide de13a was previously purified from the venom of the worm-hunting cone snail Conus delessertii from the Yucatán Channel, México. This peptide has eight cysteine (Cys) residues in the unique arrangement CCCCCCCC, which defines the cysteine framework XIII (“” represents one or more non-Cys residues). Remarkably, δ-hydroxy-lysine residues have been found only in conotoxin de13a, which also contains an unusually high proportion of hydroxylated amino acid residues. Here, we report the cDNA cloning of the complete precursor De13.1 of a related peptide, de13b, which has the same Cys framework and inter-Cys spacings as peptide de13a, and shares high protein/nucleic acid sequence identity (87%/90%) with de13a, suggesting that both peptides belong to the same conotoxin gene superfamily. Analysis of the signal peptide of precursor De13.1 reveals that this precursor belongs to a novel conotoxin gene superfamily that we chose to name gene superfamily G. Thus far superfamily G only includes two peptides, each of which contains the same, distinctive Cys framework and a high proportion of amino acid residues with hydroxylated side chains.     

Kinetic study on ESR signal decay of nitroxyl radicals,potent redox probes for in vivo ESR spectroscopy,caused by reactive oxygen species

The effect of the chemical structure of nitroxyl spin probes on the rate at which ESR signals are lost in the presence of reactive oxygen species (ROS) was examined. When the spin probes were reacted with either hydroxyl radical (OH) or superoxide anion radical (O₂⁻) in the presence of cysteine or NADH, the probes lost ESR signal depending on both their ring structure and substituents. Pyrrolidine nitroxyl probes were relatively resistant to the signal decay caused by O₂⁻ with cysteine/NADH. Signal decay rates for these reactions correlated with reported redox potentials of the nitroxyl/oxoammonium couple of spin probes, suggesting that the signal decay mechanism in both cases involves the oxidation of a nitroxyl group. The apparent rate constants of the reactions between the spin probe and OH and between the spin probe and O₂⁻ in the presence of cysteine were estimated using mannitol and superoxide dismutase (SOD), respectively, as competitive standards. The rate constants for spin probes and OH were in the order of 10⁹ M⁻¹ s⁻¹, much higher than those for the probes and O₂⁻ in the presence of cysteine (10³–10⁴ M⁻¹ s⁻¹). These basic data are useful for the measurement of OH and O₂⁻ in living animals by in vivo ESR spectroscopy.     

Rational derivation of CETP self-binding helical peptides by π-π stacking and halogen bonding: Therapeutic implication for atherosclerosis

The human cholesteryl ester transfer protein (CETP) transfers cholesteryl ester from high-density lipoprotein (HDL) to other lipoproteins and has been established as an attractive target for reducing the risk of atherosclerosis. Here, an amphipathic α-helix peptide, namely SBH-peptide (⁴⁶⁵EHLLVDFLQSLS⁴⁷⁶), was derived from the C-terminal tail of CETP. The peptide exhibits self-binding capability towards the CETP. Crystal structure analysis, molecular dynamics (MD) simulations and ab initio electron correlation characterizations of CETP–SBH-peptide complex system revealed that the Phe471 residue plays a key role in SBH-peptide binding, which can form a π-π stacking with the Phe197 residue of CETP. In addition, substitution of the hydrogen atom H4 of Phe471 with halogen atoms, in particular the bromine atom Br4, can constitute a geometrically satisfactory halogen bonding with the oxygen atom O of CETP Ile193 residue. Fluorescence polarization assays substantiated that (i) mutation of the aromatic Phe471 to small Ala residue would impair the SBH-peptide affinity with K_d change from 10.5 to 26.4 μM, indicating that the π-π stacking should exist in Phe471⋯Phe197 adduct, and (ii) substitution with Br4 can considerably improve SBH-peptide affinity by ∼3-fold, but the SBH-peptide binding does not change essentially upon substitution with Br3 (a negative control that is theoretically unable to form the halogen bonding), indicating that the rationally designed halogen bonding should form between the Phe471(Br4) residue of SBH-peptide and the Ile193 residue of CETP protein.     

Vancomycin resistance: Modeling backbone variants with d-Ala-d-Ala and d-Ala-d-Lac peptides

To seek vancomycin analogs with broader antibacterial activity, effects of backbone modifications for the agylcon 2 on binding with d-Ala-d-Ala- and d-Ala-d-Lac-containing peptides were investigated by Monte Carlo/free energy perturbation (MC/FEP) calculations. The experimental trend in binding affinities for 2 with three tripeptides was well reproduced. Possible modifications of the peptide bond between residues 4 and 5 were then considered, specifically for conversion of the OCNH linkage to CH₂NH₂⁺ (6), FCCH (7), HCCH (8), and HNCO (9). The MC/FEP results did not yield binding improvements for 7, 8, and 9, though the fluorovinyl replacement is relatively benign. The previously reported analog 6 remains as the only variant that exhibits improved affinity for the d-Ala-d-Lac sequence and acceptable affinity for the d-Ala-d-Ala sequence.     

Mechanisms and kinetic profiles of superoxide-stimulated nitrosative processes in cells using a diaminofluorescein probe

In this study, we examined the mechanisms and kinetic profiles of intracellular nitrosative processes using diaminofluorescein (DAF-2) as a target in RAW 264.7 cells. The intracellular formation of the fluorescent, nitrosated product diaminofluorescein triazol (DAFT) from both endogenous and exogenous nitric oxide (NO) was prevented by deoxygenation and by cell membrane-permeable superoxide (O₂⁻) scavengers but not by extracellular bovine Cu,Zn-SOD. In addition, the DAFT formation rate decreased in the presence of cell membrane-permeable Mn porphyrins that are known to scavenge peroxynitrite (ONOO⁻) but was enhanced by HCO₃⁻/CO₂. Together, these results indicate that nitrosative processes in RAW 264.7 cells depend on endogenous intracellular O₂⁻ and are stimulated by ONOO⁻/CO₂-derived radical oxidants. The N₂O₃ scavenger sodium azide (NaN₃) only partially attenuated the DAFT formation rate and only with high NO (>120 nM), suggesting that DAFT formation occurs by nitrosation (azide-susceptible DAFT formation) and predominantly by oxidative nitrosylation (azide-resistant DAFT formation). Interestingly, the DAFT formation rate increased linearly with NO concentrations of up to 120–140 nM but thereafter underwent a sharp transition and became insensitive to NO. This behavior indicates the sudden exhaustion of an endogenous cell substrate that reacts rapidly with NO and induces nitrosative processes, consistent with the involvement of intracellular O₂⁻. On the other hand, intracellular DAFT formation stimulated by a fixed flux of xanthine oxidase-derived extracellular O₂⁻ that also occurs by nitrosation and oxidative nitrosylation increased, peaked, and then decreased with increasing NO, as previously observed. Thus, our findings complementarily show that intra- and extracellular O₂⁻-dependent nitrosative processes occurring by the same chemical mechanisms do not necessarily depend on NO concentration and exhibit different unusual kinetic profiles with NO dynamics, depending on the biological compartment in which NO and O₂⁻ interact.     

Unlocking nature’s CH bonds

In an idealistic setting, it can be imagined that if every CH bond on an organic molecule could be selectively functionalized, the fields of chemical synthesis and drug discovery would be forever revolutionized. With the purpose of investigating the practicality of this idealistic scenario, our group has endeavored to unlock the potential of nature’s CH bonds by developing palladium-catalyzed, site selective CH insertions that can be incorporated into both known and new catalytic cycles. To this end, we have developed a number of catalytic transformations that not only provide rapid diversification of simple starting materials and natural products through CH functionalization, but streamline the synthesis of a variety of natural products with biological activity and expand upon methods to access highly valuable enantiopure materials.     

Radical scavenging and anti-inflammatory activities of (hetero)arylethenesulfonyl fluorides: Synthesis and structure-activity relationship (SAR) and QSAR studies

A series of (hetero)arylethenesulfonyl fluorides (1–58) were synthesized and screened for their in vitro antioxidant (DPPH, ABTS and DMPD methods) and anti-inflammatory activities. The results revealed that compounds 4, 15, 16, 24, 25, 26, 38, 39, 40, and 54 exhibited excellent antioxidant activity using all the three performed antioxidant methods, which were superior to the standard antioxidants ascorbic acid and gallic acid. Compounds 6–9, 11, 18, 19, 21, 22, 30, 39, 40, 44, 45, 48–50, 54, 55 and 57 displayed promising anti-inflammatory activity, which were better than the reference drug indomethacin. Preliminary structure–activity relationship (SAR) revealed that compounds containing electron donating (OH and OCH₃) groups on the phenyl ring possessed excellent antioxidant properties while compounds containing electron-withdrawing (Cl, NO₂, F and Br) groups on the phenyl ring were found to be most potent anti-inflammatory agents. The presence of SO₂F group played a crucial role in increases both antioxidant and anti-inflammatory activities.     

Impacts of sulfur regulation in vivo on arsenic accumulation and tolerance of hyperaccumulator Pteris vittata

The impacts of the regulation of sulfur (S) metabolism in vivo on arsenic (As) and S species and on As accumulation by Pteris vittata L. were investigated using a synchrotron-based X-ray-absorption fine structure method. The S assimilation inhibitor l-buthionine-sulfoximine (BSO) markedly inhibited As reduction, doubling arsenate (As(V)) content in P. vittata rhizoids. The resulting As transport blockage in rhizoids, decreased As movement to aboveground tissues by 47%. The significant impact of BSO demonstrated the vital role of sulfhydryl groups (SH) as reductants in As(V) reduction and confirmed the importance of As(V) reduction in As accumulation in this fern. The S metabolism accelerant O-acetyl-l-serine resulted in the appearance of large amounts of As–SH in rhizoids and had no obvious impact on As accumulation, but with As stress conditions, effectively increased plant biomass, possibly through chelation of excess As with SH. Thus, SH appeared able to act as both a reductant and a chelator of As in P. vittata, and the ratio of SH to As may have been a factor that determined the specific role of SH in P. vittata under these conditions.     

Antioxidant capacities in various animal sera as measured with multiple free-radical scavenging method

Scavenging abilities of animal sera against six reactive species (OH, O₂⁻, RO, t-BuOO, H₃C, and ¹O₂) were determined with the use of multiple free-radical scavenging (MULTIS) method. Commercially available sera from pig, horse, rabbit, Guinea pig, hamster and chicken were subjected to MULTIS analysis and the results were compared with human specimen. In general, animal sera showed lower scavenging ability against OH and RO radicals than human serum. However, it is noteworthy that rabbit and chicken sera have higher scavenging ability against O₂⁻ than others. This is consistent with the known data that superoxide dismutase levels in these sera are high. In addition, we determined the uric acid level in animal sera using the uricase-TOOS method. In chicken serum, uric acid was found to be the major effective component in RO scavenging. This paper is first to quantitatively evaluate antioxidant capacities in animal sera.     

Endothelial NO and O2− production rates differentially regulate oxidative,nitroxidative, and nitrosative stress in the microcirculation

Endothelial dysfunction causes an imbalance in endothelial NO and O₂⁻ production rates and increased peroxynitrite formation. Peroxynitrite and its decomposition products cause multiple deleterious effects including tyrosine nitration of proteins, superoxide dismutase (SOD) inactivation, and tissue damage. Studies have shown that peroxynitrite formation during endothelial dysfunction is strongly dependent on the NO and O₂⁻ production rates. Previous experimental and modeling studies examining the role of NO and O₂⁻ production imbalance on peroxynitrite formation showed different results in biological and synthetic systems. However, there is a lack of quantitative information about the formation and biological relevance of peroxynitrite under oxidative, nitroxidative, and nitrosative stress conditions in the microcirculation. We developed a computational biotransport model to examine the role of endothelial NO and O₂⁻ production on the complex biochemical NO and O₂⁻ interactions in the microcirculation. We also modeled the effect of variability in SOD expression and activity during oxidative stress. The results showed that peroxynitrite concentration increased with increase in either O₂⁻ to NO or NO to O₂⁻ production rate ratio (Q_O2⁻/Q_NO or Q_NO/Q_O2⁻, respectively). The peroxynitrite concentrations were similar for both production rate ratios, indicating that peroxynitrite-related nitroxidative and nitrosative stresses may be similar in endothelial dysfunction or inducible NO synthase (iNOS)-induced NO production. The endothelial peroxynitrite concentration increased with increase in both Q_O2⁻/Q_NO and Q_NO/Q_O2⁻ ratios at SOD concentrations of 0.1–100 μM. The absence of SOD may not mitigate the extent of peroxynitrite-mediated toxicity, as we predicted an insignificant increase in peroxynitrite levels beyond Q_O2⁻/Q_NO and Q_NO/Q_O2⁻ ratios of 1. The results support the experimental observations of biological systems and show that peroxynitrite formation increases with increase in either NO or O₂⁻ production, and excess NO production from iNOS or from NO donors during oxidative stress conditions does not reduce the extent of peroxynitrite mediated toxicity.     

Reactive oxygen-induced reactive oxygen formation during human sperm capacitation

Physiological processes are often activated by reactive oxygen species (ROS), such as the superoxide anion (O₂^–) and nitric oxide (NO) produced by cells. We studied the interactions between NO and O₂^–, and their generators (NO synthase, NOS, and a still elusive oxidase), in human spermatozoa during capacitation (transformations needed for acquisition of fertility). Albumin, fetal cord serum ultrafiltrate, and L-arginine triggered capacitation and ROS generation (NO and O₂^–) and superoxide dismutase (SOD) and NOS inhibitors prevented all these effects. Surprisingly, capacitation due to exogenous NO (or O₂^–) was also blocked by SOD (or NOS inhibitors). Probes used were proven specific and innocuous on spermatozoa. Whereas O₂^– was needed only for 30 min, the continuous NO generation was essential for hours. Capacitation caused a time-dependent increase in protein tyrosine nitration that was prevented by SOD and NOS inhibitors, suggesting that O₂^– and NO· also act via the formation of ONOO^–. Spermatozoa treated with NO (or O₂^–) initiated a dose-dependent O₂^– (or NO) production, providing, for the first time in cells, a strong evidence for a two-sided ROS-induced ROS generation. Data presented show a close interaction between NO and O₂^– and their generators during sperm capacitation.     

Interaction of a quercetin derivative - lensoside Aβ with liposomal membranes

Lensoside Aβ, representing the flavonol glycosides, is a compound isolated from the aerial parts of edible lentil (Lens culinaris) cultivar Tina. This substance arouses interest because so far there is very little data about secondary metabolites isolated from the leaves and stems of this plant. Additionally, bioactive potential of flavonoids is directly coupled with the membranes as a primary target of their physiological and pharmacological activity. The aim of this study was to investigate the effect of lensoside Aβ on lipid membranes. Interaction of examined compound with liposomes formed with dipalmitoylphosphatidylcholine (DPPC) was investigated with application of FTIR spectroscopy and ¹H NMR technique. Molecular localization and orientation of lensoside Aβ in a single lipid bilayer system represented by giant unilamellar vesicles, was also investigated with application of confocal fluorescence lifetime imaging microscopy (FLIM).FTIR analysis revealed that the tested compound incorporates into DPPC membranes via hydrogen bonding to lipid polar head groups in the PO₂ group region and the COPOC segment. Furthermore ¹H NMR analysis showed ordering effect in both the hydrophobic alkyl chains region and the polar heads of phospholipids. FLIM investigation has revealed roughly parallel orientation of its molecules in the membranes. This suggests that one of the possible physiological functions of this flavonol could be screening a cell against short-wavelength radiation.     

Selective protein modification by the hydroperoxide intermediate in a photoprotein,aequorin

During the course of protein modification program, we employed a recombinant aequorin, the apo-protein reconstituted with coelenterazine, and found out that the photolytic hyperperoxide modified three –S–SCH₂CHOHCHOHCH₂SH groups to –S–SCH₂CHOHCHCH–SO)H or –S–SCH₂CHOHCHCH–S(O)OH of terminal DTT connected to cysteine residues of the C¹⁴⁵, C¹⁵² and C¹⁸⁰, which turned out to locate near the chromophore.     

Geometrical effects of phospholipid olefinic bonds on the structure and dynamics of membranes: A molecular dynamics study

The trans isomers of fatty acids are found in human adipose tissue. These isomers have been linked with deleterious health effects (e.g., coronary artery disease). In this study, we performed molecular dynamics simulations to investigate the structures and dynamic properties of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) and 1-palmitoyl-2-elaidoyl sn-glycero-3-phosphatidylcholine (PEPC) lipid bilayers. The geometry of the olefinic bond and membrane packing effects significantly influenced the conformations and dynamics of the two CC single bonds adjacent to the olefinic bond. For the PEPC lipid, the two CC single bonds adjacent to the olefinic bond adopted mainly nonplanar skew–trans and planar cis–trans motifs; although the cis conformation featured relatively strong steric repulsion, it was stabilized through membrane packing because its planar structure is more suitable for membrane packing. Moreover, membrane packing effects stabilized the planar transition state for conformational conversion to a greater extent than they did with the nonplanar transition state, thereby affecting the dynamics of conformational conversion. The rotational motions of the first neighboring CC single bonds were much faster than those of typical saturated CC single bonds; in contrast, the rotational motions of the second neighboring CC single bonds were significantly slower than those of typical saturated torsion angles. The packing of PEPC lipids is superior to that of POPC lipids, leading to a smaller area per lipid, a higher order parameter and a smaller diffusion coefficient. The distinct properties of POPC and PEPC lipids result in PEPC lipids forming microdomains within a POPC matrix.     

Crystal and molecular conformation of the fluorescent probe. 8-anilino-1-naphthalenesulfonic acid (ANS).

The crystal and molecular structure of the fluorescent probe 8-anilino-1-naphthalenesulfonic acid (ANS) has been determined as the ammonium monohydrate with two conformationally distinct molecules in the triclinic $P\overline{1}$ lattice. The angles between the aromatic rings and the CNC plane are ?9/22° and 112/22° respectively. There is an NH...O intramolecular hydrogen bond in each molecule indicating that hydrogen bond formation is not dependent on the anilino geometry. There are also short intramolecular H...H contacts involving the hydrogens which have anomalous proton shifts shown in a recent NMR study.     

Influence of microbial associations on selenium localization and speciation in roots of Astragalus and Stanleya hyperaccumulators

Selenium (Se) hyperaccumulator plants can accumulate and tolerate Se up to 1% of their dry weight. Since little is known about below-ground processes of Se uptake and metabolism in hyperaccumulators, X-ray absorption spectromicroscopy was used to characterize the chemical composition and spatial distribution of Se in roots of Astragalus and Stanleya hyperaccumulators. Selenium was present throughout the roots, with the highest levels in the cortex. The main form of Se (48–95%) in both species collected from naturally seleniferous soil was an organic CSeC compound, likely methyl-selenocysteine. In addition, surprisingly high fractions (up to 35%) of elemental Se (Se⁰) were found, a form so far not reported in plants but commonly produced by Se-tolerant bacteria and fungi. Four fungi collected from hyperaccumulator roots were characterized with respect to their Se tolerance and ability to produce Se⁰, and then used to inoculate hyperaccumulators in a controlled greenhouse study. The roots of the greenhouse-grown Astragalus and Stanleya contained mainly CSeC; in most plants no Se⁰ was detected, with the exception of Astragalus nodules and roots of Astragalus inoculated with Alternaria astragali, an Se⁰-producing fungus. Apparently, Se⁰-producing endosymbionts including nitrogen-fixing bacteria and endophytic fungi or bacteria in the root can affect Se speciation in hyperaccumulator roots. Microbes that affect plant Se speciation may be applicable in phytoremediation and biofortification, especially if they are promiscuous and affect Se tolerance in crop species.