Effects of hydrophobicity and anions on self‐assembly of the peptide EMK16‐II

Effects of hydrophobic and electrostatic interactions on the self‐assembling process of the ionic‐complementary peptide EMK16‐II are investigated by atomic force microscopy imaging, circular dichroism spectra, light scattering, and chromatography. It is found that the hydrophobicity of the peptide promotes the aggregation in pure water even at a very low concentration, resulting in a much lower critical aggregation concentration than that of another peptide, EAK16‐II. The effect of anions in solution with different valences on electrostatic interactions is also important. Monovalent anions (Cl^? and Ac^?) with a proper concentration can facilitate the formation of peptide fibrils, with Cl^? of smaller size being more effective than Ac^? of larger size. However, only small amounts of fibrils, but plenty of large amorphous aggregates, are found when the peptide solution is incubated with multivalent anions, such as SO, C₆H₅O, and HPO. More importantly, by gel filtration chromatography, the citrate anion, which induces a similar effect on the self‐assembling process of EMK16‐II as that of SO and HPO, can interact with two or more positively charged residues of the peptide and reside in the amorphous aggregates. This implies a “salt bridge” effect of multivalent anions on the peptide self‐assembling process, which can interpret a previous puzzle why divalent cations inhibit the formation of ordered nanofibrils of the ionic‐complementary peptides. Thus, our results clarify the important effects of hydrophobic and electrostatic interactions on the self‐assembling process of the ionic‐complementary peptides. These are greatly helpful for us to understand the mechanism of peptides' self‐assembling process and protein folding and aggregation. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 318–329, 2010. This article was originally published online as an acceptedpreprint. The “Published Online” date corresponds to the preprintversion. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Characterization of site‐directed mutants of residues R58, R59, D116, W340 and R372 in the active site of E. coli cystathionine β‐lyase

Cystathionine β‐lyase (CBL) catalyzes the hydrolysis of L ‐cystathionine (L ‐Cth) to produce L ‐homocysteine, pyruvate, and ammonia. A series of active‐site mutants of Escherichia coli CBL (eCBL) was constructed to investigate the roles of residues R58, R59, D116, W340, and R372 in catalysis and inhibition by aminoethoxyvinylglycine (AVG). The effects of these mutations on the k_cat/K for the β‐elimination reaction range from a reduction of only 3‐fold for D116A and D116N to 6 orders of magnitude for the R372L and R372A mutants. The order of importance of these residues for the hydrolysis of L ‐Cth is: R372 >> R58 > W340 ≈ R59 > D116. Comparison of the kinetic parameters for L ‐Cth hydrolysis with those for inhibition of eCBL by AVG demonstrates that residue R58 tethers the distal carboxylate group of the substrate and confirms that residues W340 and R372 interact with the α‐carboxylate moiety. The increase in the pK_a of the acidic limb and decrease in the pK_a of the basic limb of the k_cat/K versus pH profiles of the R58K and R58A mutants, respectively, support a role for this residue in modulating the pK_a of an active‐site residue.     

Exploration of the active site of Escherichia coli cystathionine γ‐synthase

Cystathionine γ‐synthase (CGS) catalyzes the condensation of O‐succinyl‐L ‐homoserine (L ‐OSHS) and L ‐cysteine (L ‐Cys), to produce L ‐cystathionine (L ‐Cth) and succinate, in the first step of the bacterial transsulfuration pathway. In the absence of L ‐Cys, the enzyme catalyzes the futile α,γ‐elimination of L ‐OSHS, yielding succinate, α‐ketobutyrate, and ammonia. A series of 16 site‐directed variants of Escherichia coli CGS (eCGS) was constructed to probe the roles of active‐site residues D45, Y46, R48, R49, Y101, R106, N227, E325, S326, and R361. The effects of these substitutions on the catalytic efficiency of the α,γ‐elimination reaction range from a reduction of only ～2‐fold for R49K and the E325A,Q variants to 310‐ and 760‐fold for R361K and R48K, respectively. A similar trend is observed for the k_cat/K of the physiological, α,γ‐replacement reaction. The results of this study suggest that the arginine residues at positions 48, 106 and 361 of eCGS, conserved in bacterial CGS sequences, tether the distal and α‐carboxylate moieties, respectively, of the L ‐OSHS substrate. In contrast, with the exception of the 13‐fold increase observed for R106A, the K is not markedly affected by the site‐directed replacement of the residues investigated. The decrease in k_cat observed for the S326A variant reflects the role of this residue in tethering the side chain of K198, the catalytic base. Although no structures exist of eCGS bound to active‐site ligands, the roles of individual residues is consistent with the structures inhibitor complexes of related enzymes. Substitution of D45, E325, or Y101 enables a minor transamination activity for the substrate L ‐Ala.     

Hydroxyl and superoxide radical scavenging abilities of chromonyl‐thiazolidine‐2,4‐dione compounds

The oxygen free radical scavenging activities of 15 chromonyl‐thiazolidine‐2,4‐dione compounds (CTDs) were examined in chemical systems producing superoxide anion radicals, O (potasium superoxide–18‐crown‐6 ether–DMSO), and hydroxyl radicals, HO^? (a Fenton reaction: Fe(II)–H₂O₂–sodium trifluoroacetate, pH 6.15). Chemiluminescence and electron spin resonance (ESR) spectroscopy using 5,5‐dimethyl‐1‐pyrroline‐1‐oxide (DMPO) as spin trap were applied to evaluate antioxidant behaviour of CTDs towards the oxygen radicals. The results indicated that 11 of the 15 tested compounds showed a significant inhibitory effect on the chemiluminescence generated from the O‐generating system, ranging from 41 to 86%, and 13 CTDs quenched the ESR signal of the DMPO–OH spin adduct by 33–86%, at a concentration of 1 mmol L^?1. Our findings demonstrate that CTDs could be good free radical scavengers. Copyright © 2008 John Wiley & Sons, Ltd.     

Scavenging of superoxide anion radical and hydroxyl radical by novel thiazolyl‐thiazolidine‐2,4‐dione compounds

The antioxidant behavior of a series of new synthesized substituted thiazolyl‐thiazolidine‐2,4‐dione compounds (TZDs) was examined using chemiluminescence and electron paramagnetic resonance spin trapping techniques. 5,5‐Dimethyl‐1‐pyrroline‐N‐oxide (DMPO) was used as the spin trap. The reactivity of TZDs with superoxide anion radical (O) and hydroxyl radical (HO^?) was evaluated using potassium superoxide/18‐crown‐6 ether dissolved in dimethylsulfoxide, and the Fenton‐like reaction (Fe²⁺ + H₂O₂), respectively. The results showed that TZDs efficiently inhibited light emission from the O generating system at a concentration of 0.05–1 mmol L^?1 (5–94% reductions were found at 1 mmol L^?1 concentration). The TZD compounds showed inhibition of HO^?‐dependent DMPO–OH spin adduct formation from DMPO (the amplitude decrease ranged from 8 to 82% at 1 mmol L^?1 concentration). The findings showed that examined TZDs had effective activities as radical scavengers. Copyright © 2009 John Wiley & Sons, Ltd.     

Hemoglobin induces the expression of indoleamine 2,3‐dioxygenase in dendritic cells through the activation of PI3K,PKC, and NF‐κB and the generation of reactive oxygen species

Indoleamine 2,3‐dioxygenase (IDO) is the rate‐limiting enzyme in the kynurenine (Kyn) pathway of tryptophan (Trp) metabolism. IDO is immunosuppressive and is induced by inflammation in macrophages and dendritic cells (DCs). Previous studies have shown the serum Kyn/Trp levels in patients with hemolytic anemia to be notably high. In the present study, we demonstrated that hemoglobin (Hb), but not hemin or heme‐free globin (Apo Hb), induced IDO expression in bone marrow‐derived myeloid DCs (BMDCs). Hb induced the phosphorylation and degradation of IκBα. Hb‐induced IDO expression was inhibited by inhibitors of PI3‐kinase (PI3K), PKC and nuclear factor (NF)‐κB. Hb translocated both RelA and p52 from the cytosol to the nucleus and induced the intracellular generation of reactive oxygen species (ROS). Hb‐induced IDO expression was inhibited by anti‐oxidant N‐acetyl‐L ‐cysteine (NAC) or mixtures of SOD and catalase, however, IDO expression was enhanced by 3‐amino‐1,2,4‐triazole, an inhibitor of catalase, suggesting that the generation of ROS such as O, H₂O₂, and hydroxyl radical is required for the induction of IDO expression. The generation of ROS was inhibited by a PKC inhibitor, and this action was further enhanced by addition of a PI3K inhibitor. Hb induced Akt phosphorylation, which was inhibited by a PI3K inhibitor and enhanced by a PKC inhibitor. These results suggest that the activation of NF‐κB through the PI3K‐PKC‐ROS and PI3K‐Akt pathways is required for the Hb‐induced IDO expression in BMDCs. J. Cell. Biochem. 108: 716–725, 2009. © 2009 Wiley‐Liss, Inc.     

Determination of enantiomerization barriers of hypericin and pseudohypericin by dynamic high‐performance liquid chromatography on immobilized polysaccharide‐type chiral stationary phases and off‐column racemization experiments

Direct enantiomer separation of hypericin, pseudohypericin, and protohypericin was accomplished by high‐performance liquid chromatography (HPLC) using immobilized polysaccharide‐type chiral stationary phases (CSPs). Enantioselectivities up to 1.30 were obtained in the polar‐organic elution mode whereby for hypericin and pseudohypericin Chiralpak IC [chiral selector being cellulose tris(3,5‐dichlorophenylcarbamate)] and for protohypericin Chiralpak IA (chiral selector being the 3,5‐dimethylphenylcarbamate of amylose) gave favorable results. Enantiomers were distinguished by on‐line electronic circular dichroism detection. Optimized enantioselective chromatographic conditions were the basis for determining stereodynamic parameters of the enantiomer interconversion process of hypericin and pseudohypericin. Rate constants delivered by computational simulation of dynamic HPLC elution profiles (stochastic model, consideration of peak tailing) were used to calculate averaged enantiomerization barriers (ΔG) of 97.6–99.6 kJ/mol for both compounds (investigated temperature range 25–45°C). Complementary variable temperature off‐column (i.e., in solution) racemization experiments delivered ΔG = 97.1–98.0 kJ/mol (27–45°C) for hypericin and ΔG = 98.9–101.4 kJ/mol (25–55°C) for pseudohypericin. An activation enthalpy of ΔH^# = 86.0 kJ/mol and an activation entropy of ΔS^# = ?37.7 J/(K mol) were calculated from hypericin racemization kinetics in solution, whereas for pseudohypericin these figures amounted to 74.1 kJ/mol and ?82.6 J/(K mol), respectively. Although the natural phenanthroperylene quinone pigments hypericin and pseudohypericin as well as their biological precursor protohypericin are chiral and can be separated by enantioselective HPLC low enantiomerization barriers seem to prevent the occurrence of an excess of one enantiomer under typical physiological conditions—at least as long as stereoselective intermolecular interactions with other chiral entities are absent. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Scavenging capacities of some thiazolyl thiazolidine‐2,4‐dione compounds on superoxide radical,hydroxyl radical,and DPPH radical

The scavenging effects of eighteen thiazolyl thiazolidine‐2,4‐dione compounds (TTCs) on superoxide radical , hydroxyl radical HO^?, and 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH^?) radical were evaluated by the chemiluminescence technique, electron spin resonance spectrometry (ESR) and visible spectrophotometry, respectively. The examined compounds were shown to have 27–59% scavenging ability, 19–69% HO^? scavenging activity and 2–32% DPPH^? scavenging ability. This property of the tested compound seems to be important in the prevention of various diseases of free radicals etiology. Copyright © 2009 John Wiley & Sons, Ltd.     

Evaluating four mathematical models for nitrous oxide production by autotrophic ammonia‐oxidizing bacteria

There is increasing evidence showing that ammonia‐oxidizing bacteria (AOB) are major contributors to N₂O emissions from wastewater treatment plants (WWTPs). Although the fundamental metabolic pathways for N₂O production by AOB are now coming to light, the mechanisms responsible for N₂O production by AOB in WWTP are not fully understood. Mathematical modeling provides a means for testing hypotheses related to mechanisms and triggers for N₂O emissions in WWTP, and can then also become a tool to support the development of mitigation strategies. This study examined the ability of four mathematical model structures to describe two distinct mechanisms of N₂O production by AOB. The production mechanisms evaluated are (1) N₂O as the final product of nitrifier denitrification with NO as the terminal electron acceptor and (2) N₂O as a byproduct of incomplete oxidation of hydroxylamine (NH₂OH) to NO. The four models were compared based on their ability to predict N₂O dynamics observed in three mixed culture studies. Short‐term batch experimental data were employed to examine model assumptions related to the effects of (1) NH concentration variations, (2) dissolved oxygen (DO) variations, (3) NO accumulations and (4) NH₂OH as an externally provided substrate. The modeling results demonstrate that all these models can generally describe the NH, NO, and NO data. However, none of these models were able to reproduce all measured N₂O data. The results suggest that both the denitrification and NH₂OH pathways may be involved in N₂O production and could be kinetically linked by a competition for intracellular reducing equivalents. A unified model capturing both mechanisms and their potential interactions needs to be developed with consideration of physiological complexity. Biotechnol. Bioeng. 2013; 110: 153–163. © 2012 Wiley Periodicals, Inc.     

Ground‐ and excited‐state proton transfer and antioxidant activity of 3‐hydroxyflavone in egg yolk phosphatidylcholine liposomes: absorption and fluorescence spectroscopic studies

Excited‐state intramolecular proton transfer (ESIPT) and dual luminescence behaviour of 3‐hydroxyflavone (3‐HF) have been utilized to monitor its binding to liposomal membranes prepared from egg yolk phosphatydilcholine (EYPC). Additionally, absorption spectrophotometric assay has been performed to evaluate the antioxidant activity of 3‐HF against lipid peroxidation in this membrane system. When 3‐HF molecules are partitioned into EYPC liposomes, a weak long‐wavelength absorption band with λ ～410 nm appears in addition to the principal absorption at ～λ = 345 nm. Selective excitation of the 410 nm band produces the characteristic emission (λ～460 nm) of the ground‐state anionic species, whereas excitation at the higher energy absorption band leads to dual emission with predominatly ESIPT tautomer fluorescence (λ = 528 nm). Both ESIPT tautomer and the anionic species exhibit fairly high fluorescence anisotropy (r) values (r = 0.122 and 0.180, respectively). Biexponential fluorescence decay kinetics are observed for the ESIPT tautomer as well as the ground‐state anionic forms, indicating heterogeneity in the microenvironments of the corresponding emitting species. Furthermore, we demonstrate that lipid peroxidation of EYPC liposomes is significantly inhibited upon 3‐HF binding, suggesting that 3‐HF can be potentially useful as an inhibitor of peroxidative damage of cell membranes. Copyright © 2008 John Wiley & Sons, Ltd.     

NMR structure of duplex DNA containing the α‐OH‐PdG·dA base pair: A mutagenic intermediate of acrolein

Acrolein, a cell metabolic product and main component of cigarette smoke, reacts with DNA generating α‐OH‐PdG lesions, which have the ability to pair with dATP during replication thereby causing G to T transversions. We describe the solution structure of an 11‐mer DNA duplex containing the mutagenic α‐OH‐PdG·dA base pair intermediate, as determined by solution nuclear magnetic resonance (NMR) spectroscopy and retrained molecular dynamics (MD) simulations. The NMR data support a mostly regular right‐handed helix that is only perturbed at its center by the presence of the lesion. Undamaged residues of the duplex are in anti orientation, forming standard Watson‐Crick base pairs alignments. Duplication of proton signals at and near the damaged base pair reveals the presence of two enantiomeric duplexes, thus establishing the exocyclic nature of the lesion. The α‐OH‐PdG adduct assumes a syn conformation pairing to its partner dA base that is protonated at pH 6.6. The three‐dimensional structure obtained by restrained molecular dynamics simulations show hydrogen bond interactions that stabilize α‐OH‐PdG in a syn conformation and across the lesion containing base pair. We discuss the implications of the structures for the mutagenic bypass of acrolein lesions. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 391–401, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Small‐molecule reductants inhibit multicatalytic activity of AA‐NADase from Agkistrodon acutus venom by reducing the disulfide‐bonds and Cu(II) of enzyme

AA‐NADase from Agkistrodon acutus venom is a unique multicatalytic enzyme with both NADase and AT(D)Pase activities. Among all identified NADases, only AA‐NADase contains Cu(II) and has disulfide‐bond linkages between two peptide chains. The effects of the reduction of the disulfide‐bonds and Cu(II) in AA‐NADase by small‐molecule reductants on its NADase and ADPase activities have been investigated by polyacrylamide gel electrophoresis, high performance liquid chromatography, electron paramagnetic resonance spectroscopy and isothermal titration calorimetry. The results show that AA‐NADase has six disulfide‐bonds and fifteen free cysteine residues. L‐ascorbate inhibits AA‐NADase on both NADase and ADPase activities through the reduction of Cu(II) in AA‐NADase to Cu(I), while other reductants, dithiothreitol, glutathione and tris(2‐carboxyethyl)phosphine inhibit both NADase and ADPase activities through the reduction of Cu(II) to Cu(I) and the cleavage of disulfide‐bonds in AA‐NADase. Apo‐AA‐NADase can recover its NADase and ADPase activities in the presence of 1 mM Zn(II). However, apo‐AA‐NADase does not recover any NADase or ADPase activity in the presence of 1 mM Zn(II) and 2 mM TCEP. The multicatalytic activity relies on both disulfide‐bonds and Cu(II), while Cu(I) can not activate the enzyme activities. AA‐NADase is probably only active as a dimer. The inhibition curves for both ADPase and NADase activities by each reductant share a similar trend, suggesting both ADPase and NADase activities probably occur at the same site. In addition, we also find that glutathione and L‐ascorbate are endogenous inhibitors to the multicatalytic activity of AA‐NADase. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 141–149, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Heating‐induced conformational change of a novel β‐(1→3)‐D‐glucan from Pleurotus geestanus

Recently, we isolated and purified a neutral polysaccharide (PGN) from edible fungus Pleurotus geestanus. Its structure was characterized by a range of physical–chemical methods, including high performance anion exchange chromatography, uronic acid, and protein analyses, size exclusion chromatography with ultraviolet, refractive index and light scattering detectors, and nuclear magnetic resonance. Our results revealed that PGN is a novel β‐(1→3)‐D ‐glucan with glucose attached to every other sugar residues at Position 6 in the backbone. It has a degree of branching of 1/2. Such structure is different from typical β‐(1→3)‐D ‐glucans schizophyllan and lentinan in which DB is 1/3 and 2/5, respectively. Rheological study showed a very interesting melting behavior of PGN in water solution: heating PGN in water leads to two transitions, in the range of 8–12.5°C and 25–60°C, respectively. The melting behavior and conformational changes were characterized by rheometry, micro‐differential scan calorimetry, atomic force microscopy, static and dynamic light scattering at different temperatures. The first heating‐induced transition corresponds to the disintegration of polymer bundles into small helical clusters, resembling the heating‐induced dissociation of SPG in water at 7°C; the second one might correspond to the dissociation of helical strands to individual chains. The ability of PGN to undergo a conformation/viscosity transition in water upon heating is very valuable to immobilize cells or enzymes or therapeutic DNA/RNA, which makes PGN a potentially useful biomaterial. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 121–131, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Kinetics of the self‐aggregation and film formation of poly‐L‐proline at high temperatures explored by circular dichroism spectroscopy

Poly‐L ‐proline has been used as a model system for various purposes over a period of more than 60 years. Its relevance among the protein/peptide community stems from its use as a reference system for determining the conformational distributions of unfolded peptides and proteins, its use as a molecular ruler, and from the pivotal role of proline residues in conformational transitions and protein–protein interactions. While several studies indicate that polyproline can aggregate and precipitate in aqueous solution, a systematic study of the aggregation process is still outstanding. We found, by means of UV‐circular dichroism and IR measurements, that polyproline is predominately monomeric at room temperature at millimolar concentrations. Upon heating, the polypeptide stays in its monomeric state until the temperature reaches a threshold of ca. 60°C. At higher temperatures, the peptide aggregates as a film on the inside surface of the employed cuvette. The process proceeds on a time scale of 10³ s and can best be described by a bi‐exponential relaxation function. The respective CD and IR spectra are qualitatively different from the canonical spectra of polyproline in aqueous solution, and are indicative of a highly packed state. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 451–457, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Dynamic and structural scalings of the complexation between pDNA and bPEI in semidilute and low‐salt solutions

Using a combination of static and dynamic laser light scattering, we investigated the complexation of a supercoiled plasmid DNA (pDNA, 10⁴ bp) and a branched polyethyleneimine (bPEI, M_w = 25 kD) in semidilute and low‐salt aqueous solutions. Our results unearth some scaling laws for dynamic and structural properties of the resultant complexes (polyplexes) with different bPEI:pDNA (N:P) molar ratios. Namely, the average scattering intensity (<I>) and the average linewidth of the Rayleigh peak (<Γ>) are scaled to the scattering vector (q) as <I> ∝ q or <Γ> ∝ q, where α_S and α_D are two N:P dependent scaling exponents. The N:P ratio strongly affects the complexation. When N:P < 2.0, the motions of the negatively charged and extended pDNA chains and the polyplexes are highly correlated so that they behave like a transient network with a fractal dimension. As the N:P ratio increases, nearly all of pDNA chains condensed and the overall charge of the polyplexes reverses to slightly positive, resulting in a turbid dispersion of large loose aggregates made of smaller, but more compact, polyplexes. Further increase of N:P finally disrupts large loose aggregates, leading to a homogeneous transparent dispersion of the polyplexes. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 571–577, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Intracellular chloride and calcium transients evoked by γ‐aminobutyric acid and glycine in neurons of the rat inferior colliculus

Microfluorometric recordings showed that the inhibitory neurotransmitters γ‐aminobutyric acid (GABA) and glycine activated transient increases in the intracellular Cl⁻ concentration in neurons of the inferior colliculus (IC) from acutely isolated slices of the rat auditory midbrain. Current recordings in gramicidin‐perforated patch mode disclosed that GABA and glycine mainly evoked inward or biphasic currents. These currents were dependent on HCO and characterized by a continuous shift of their reversal potential (E_GABA/gly) in the positive direction. In HCO‐buffered saline, GABA and glycine could also evoke an increase in the intracellular Ca²⁺ concentration. Ca²⁺ transients occurred only with large depolarizations and were blocked by Cd²⁺, suggesting an activation of voltage‐gated Ca²⁺ channels. However, in the absence of HCO, only a small rise, if any, in the intracellular Ca²⁺ concentration could be evoked by GABA or glycine. We suggest that the activation of GABA_A or glycine receptors results in an acute accumulation of Cl⁻ that is enhanced by the depolarization owing to HCO efflux, thus shifting E_GABA/gly to more positive values. A subsequent activation of these receptors would result in a strenghtened depolarization and an enlarged Ca²⁺ influx that might play a role in the stabilization of inhibitory synapses in the auditory pathway. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 386–396, 1999     

Impact of α‐hydroxy‐propanodeoxyguanine adducts on DNA duplex energetics: Opposite base modulation and implications for mutagenicity and genotoxicity

Acrolein is an α,β‐unsaturated aldehyde that is a major environmental pollutant, as well as a product of cellular metabolism. DNA bases react with acrolein to form two regioisomeric exocyclic guanine adducts, namely γ‐hydroxy‐propanodeoxyguanosine (γ‐OH‐PdG) and its positional isomer α‐hydroxy‐propanodeoxyguanosine (α‐OH‐PdG). The γ‐OH‐PdG isomer adopts a ring‐opened conformation with minimal structural perturbation of the DNA host duplex. Conversely, the α‐OH‐PdG isomer assumes a ring‐closed conformation that significantly disrupts Watson‐Crick base‐pair alignments within the immediate vicinity of the damaged site. We have employed a combination of calorimetric and spectroscopic techniques to characterize the thermodynamic origins of these lesion‐induced structural alterations. Specifically, we have assessed the energetic impact of α‐OH‐PdG centered within an 11‐mer duplex by hybridizing the adduct‐containing oligonucleotide with its complementary strand harboring a central base N [where N = C or A], yielding a pair of duplexes containing the nascent lesion (α‐OH‐PdG·C) or mismatched adduct (α‐OH‐PdG·A), respectively. Our data reveal that the nascent lesion is highly destabilizing, whereas its mismatched counterpart partially ameliorates α‐OH‐PdG‐induced destabilization. Collectively, our data provide energetic characterizations of the driving forces that modulate error‐free versus error‐prone DNA translesion synthesis. The biological implications of our findings are discussed in terms of energetically probing acrolein‐mediated mutagenicity versus adduct‐induced genotoxicity. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 370–382, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Crystal structure of an S‐formylglutathione hydrolase from Pseudoalteromonas haloplanktis TAC125

S‐formylglutathione hydrolases (FGHs) constitute a family of ubiquitous enzymes which play a key role in formaldehyde detoxification both in prokaryotes and eukaryotes, catalyzing the hydrolysis of S‐formylglutathione to formic acid and glutathione. While a large number of functional studies have been reported on these enzymes, few structural studies have so far been carried out. In this article we report on the functional and structural characterization of PhEst, a FGH isolated from the psychrophilic bacterium Pseudoalteromonas haloplanktis. According to our functional studies, this enzyme is able to efficiently hydrolyze several thioester substrates with very small acyl moieties. By contrast, the enzyme shows no activity toward substrates with bulky acyl groups. These data are in line with structural studies which highlight for this enzyme a very narrow acyl‐binding pocket in a typical α/β‐hydrolase fold. PhEst represents the first cold‐adapted FGH structurally characterized to date; comparison with its mesophilic counterparts of known three‐dimensional structure allowed to obtain useful insights into molecular determinants responsible for the ability of this psychrophilic enzyme to work at low temperature. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 669–677, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Effect of one D‐Leu residue on right‐handed helical ‐L‐Leu‐Aib‐ peptides in the crystal state

Four diastereomeric‐Leu‐Leu‐Aib‐Leu‐Leu‐Aib‐peptides, Boc‐D ‐Leu‐L ‐Leu‐Aib‐L ‐Leu‐L ‐Leu‐Aib‐OMe (1), Boc‐L ‐Leu‐D ‐Leu‐Aib‐L ‐Leu‐L ‐Leu‐Aib‐OMe (2), Boc‐L ‐Leu‐L ‐Leu‐Aib‐D ‐Leu‐L ‐Leu‐Aib‐OMe (3), and Boc‐L ‐Leu‐L ‐Leu‐Aib‐L ‐Leu‐D ‐Leu‐Aib‐OMe (4), were synthesized. The crystals of the four hexapeptides were characterized by X‐ray crystallographic analysis. Two diastereomeric hexapeptides 1 and 2 having D ‐Leu(1) or D ‐Leu(2) were folded into right‐handed (P) 3 ₁₀ ‐helical structures, while peptide 3 having D ‐Leu(4) was folded into a turn structure nucleated by type III′ and I$' \bf{\beta}$ ‐turns, and peptide 4 having D ‐Leu(5) was folded into a left‐handed (M) 3 ₁₀ ‐helical structure. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.