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BBA - Proteins and Proteomics (v.1814, #12)

Editorial Board (pp. i).

The reducing component BoxA of benzoyl-coenzyme A epoxidase from Azoarcus evansii is a [4Fe–4S] protein by Liv J. Rather; Eckhard Bill; Wael Ismail; Georg Fuchs (pp. 1609-1615).

BoxA is the reductase component of the benzoyl-coenzyme A (CoA) oxidizing epoxidase enzyme system BoxAB. The enzyme catalyzes the key step of an hitherto unknown aerobic, CoA-dependent pathway of benzoate metabolism, which is the epoxidation of benzoyl-CoA to the non-aromatic 2,3-epoxybenzoyl-CoA. The function of BoxA is the transfer of two electrons from NADPH to the epoxidase component BoxB. We could show recently that BoxB is a diiron enzyme, whereas here we demonstrate that BoxA harbors an FAD and two [4Fe–4S] clusters per protein monomer. The characterization of BoxA was hampered by severe oxygen sensitivity; the cubane [4Fe–4S] clusters degrade already with traces of oxygen. Interestingly, the adventitiously formed [3Fe–4S] centers could be reconstituted in vitro by adding Fe(II) and sulfide to retrieve the native cubane centers. BoxA is the first example of a reductase of this type that has an FAD and two bacterial ferredoxin-type [4Fe–4S] clusters. In other cases within the catalytically versatile family of diiron enzymes, the related reductases have plant-type ferredoxin or Rieske-type [2Fe–2S] centers only.► BoxA acts as reductase in the benzoyl-CoA oxidizing epoxidase enzyme system BoxAB. ► Homodimeric BoxA harbors an FAD and two [4Fe–4S] clusters per protein monomer. ► The [4Fe–4S] clusters degrade with traces of O₂, but could be reconstituted in vitro. ► BoxAB(C) represent a new subclass of bacterial multicomponent monooxygenases (BMO). ► Compared to BMOs, BoxA is the only dimeric reductase with FAD and [4Fe–4S] centers.

Keywords: Abbreviations; CoA; coenzyme A; box; benzoate oxidation; ICP-OES; Inductively Coupled Plasma-Optical Emission Spectrometry; EPR; electron paramagnetic resonance; DTE; dithioerythritolEPR; Mössbauer; Reductase; Benzoate; Metabolism; Bacterial multicomponent monooxygenase

The reducing component BoxA of benzoyl-coenzyme A epoxidase from Azoarcus evansii is a [4Fe–4S] protein by Liv J. Rather; Eckhard Bill; Wael Ismail; Georg Fuchs (pp. 1609-1615).

Single mutations outside the active site affect the substrate specificity in a β-glycosidase by Mendonca Lúcio M.F. Mendonça; Sandro R. Marana (pp. 1616-1623).

A library of random mutants of the β-glycosidase Sfβgly was screened for mutations that affect its specificity for the substrate glycone (β-d-fucoside versus β-d-glucoside). Among mutations selected (T35A, R189G, Y345C, P348L, S358F, S378G, N400D, S424F, F460L, and R474H), eight occurred in the C-terminal half of Sfβgly and only two were at the active site (R189G and N400D). Tryptophan fluorescence spectra and thermal inactivation showed that the selected mutants and wild-type Sfβgly are similarly folded. Enzyme kinetics confirmed that these mutations resulted in broadening or narrowing of the preference for the substrate glycone. Structural modeling and interaction maps revealed contact pathways that connect the sites of the selected mutations through up to three interactions to the active site residues E399, W444, and E187, which are involved in substrate binding and catalysis. Interestingly, independently selected mutations (Y345C, P348L, and R189G; S424F and N400D) were placed on the same contact pathway. Moreover, ( k_cat/ K_m fucoside)/( k_cat/ K_m glucoside) ratios showed that mutations at intermediate residues of the same contact pathway often had similar effects on substrate specificity. Finally mutations in the same contact pathway caused similar structural disturbance as evidenced by acrylamide quenching of the Sfβgly fluorescence. Based on these data, it is proposed that the effects of the selected mutations were propagated into the active site through groups of interacting residues (contact pathways) changing the Sfβgly substrate specificity.► We selected single mutations altering the substrate specificity of a β-glycosidase ► These mutations are far from the active site and concentrated in the C-terminal half ► These mutations are inserted in contact networks that connect to the active site ► Mutations on the same network have similar effects on the specificity and structure ► We propose that mutation effects may reach the active site via contact networks

Keywords: Abbreviations; Sfβgly; β-glycosidase from; Spodoptera frugiperda; NPβfuc; p; -nitrophenyl β-; d; -fucoside; NPβgal; p; -nitrophenyl β-; d; -galactoside; NPβglc; p; -nitrophenyl β-; d; -glucosideβ-glucosidase; Glycoside hydrolase; Substrate specificity; Contact pathways

Single mutations outside the active site affect the substrate specificity in a β-glycosidase by Mendonca Lúcio M.F. Mendonça; Sandro R. Marana (pp. 1616-1623).

Human inter-α-inhibitor is a substrate for factor XIIIa and tissue transglutaminase by Carsten Scavenius; Kristian W. Sanggaard; Camilla L. Nikolajsen; Steffen Bak; Zuzana Valnickova; Thogersen Ida B. Thøgersen; Ole N. Jensen; Hojrup Peter Højrup; Jan J. Enghild (pp. 1624-1630).

In this study, we show that inter-α-inhibitor is a substrate for both factor XIIIa and tissue transglutaminase. These enzymes catalyze the incorporation of dansylcadaverine and biotin–pentylamine, revealing that inter-α-inhibitor contains reactive Gln residues within all three subunits. These findings suggest that transglutaminases catalyze the covalent conjugation of inter-α-inhibitor to other proteins. This was demonstrated by the cross-linking between inter-α-inhibitor and fibrinogen by either factor XIIIa or tissue transglutaminase. Finally, using quantitative mass spectrometry, we show that inter-α-inhibitor is cross-linked to the fibrin clot in a 1:20 ratio relative to the known factor XIIIa substrate α2-antiplasmin. This interaction may protect fibrin or other Lys-donating proteins from adventitious proteolysis by increasing the local concentration of bikunin. In addition, the reaction may influence the TSG-6/heavy Chain 2-mediated transfer of heavy chains observed during inflammation.► Inter-α-inhibitor is a substrate for factor XIIIa and tissue transglutaminase. ► We demonstrate cross-linking of inter-α-inhibitor and fibrinogen by factor XIIIa. ► Inter-α-inhibitor is cross-linked to the plasma clot.

Keywords: Abbreviations; IαI; Inter-α-inhibitor; HC; Heavy Chain; TSG-6; Tumor necrosis factor stimulated gene-6 protein; FXIIIa; Activated Factor XIII A subunit; FXIII; Factor XIII zymogen; TTG; tissue transglutaminase; ChonABC; Chondroitinase ABC; α2AP; α2-antiplasminInter-α-inhibitor; Proteoglycan; Transglutaminase; Extracellular matrix

In-situ confocal Raman observation of structural changes of insulin crystals in sequential dehydration process by Guang Zeng; Jing-Jing Shou; Kai-Kai Li; Yun-Hong Zhang (pp. 1631-1640).

In-situ confocal Raman spectroscopy combined with relative humidity (RH) control technique was used to study the sequential dehydration process of insulin crystals. By gradually decreasing the ambient RH of the insulin crystal, the content of the hydration water in the crystal was quantitatively controlled. Tyrosine (Tyr) residues were very sensitive to the micro-environmental changes, and four Raman features 828cm⁻¹, 852cm⁻¹, 1174cm⁻¹ and 1206cm⁻¹ of Tyr were employed to monitor the dehydration process. Taking advantage of the ratios I₈₅₂/ I₈₂₈ at different RH values, the mole fractions of the ‘exposed’ and ‘buried’ Tyr residues were estimated. Moreover, using the ratio I₁₁₇₄/ I₁₂₀₆ as an indicator of the dehydration process, three RH regions were discriminated. This is believed to imply that different types of the hydration water were lost step by step, i.e. firstly the ‘second-layer’ and ‘first-layer’ classes, then the ‘contact’ class, and finally, the ‘inside’ class. In addition, the profile of the amide I band was observed to gradually change with RH. By band fitting of the amide I region, changes in secondary structure were quantitatively determined. And the results showed that nearly 17% of α-helix converted into β-sheet with RH decreasing from 92% to 2%.► The sequential dehydration process of insulin crystals has been investigated. ► Different kinds of hydration water were observed to be lost step by step. ► The hydration water significantly affects the environments of Tyr residues. ► The loss of the hydration water leads to changes in the secondary structure.

Keywords: Abbreviations; RH; relative humidity; Tyr; tyrosine; Trp; tryptophan; SNR; signal to noise ratio; Phe; phenylalanine; Cys; cystine; FWHM; full width at half maximum; SAS; solvent accessible surfaceRaman spectroscopy; Insulin; Dehydration; Secondary structure

In-situ confocal Raman observation of structural changes of insulin crystals in sequential dehydration process by Guang Zeng; Jing-Jing Shou; Kai-Kai Li; Yun-Hong Zhang (pp. 1631-1640).

The crystal structure of ADP-L- glycero-D- manno-heptose-6-epimerase (HP0859) from Helicobacter pylori by Md Munan Shaik; Giuseppe Zanotti; Laura Cendron (pp. 1641-1647).

Helicobacter pylori, the human pathogen that affects about half of the world population and that is responsible for gastritis, gastric ulcer and adenocarcinoma and MALT lymphoma, owes much of the integrity of its outer membrane on lipopolysaccharides (LPSs). Together with their essential structural role, LPSs contribute to the bacterial adherence properties, as well as they are well characterized for the capability to modulate the immuno-response. In H. pylori the core oligosaccharide, one of the three main domains of LPSs, shows a peculiar structure in the branching organization of the repeating units, which displayed further variability when different strains have been compared. We present here the crystal structure of ADP-L- glycero-D- manno-heptose-6-epimerase (HP0859, rfaD), the last enzyme in the pathway that produces L- glycero-D- manno-heptose starting from sedoheptulose-7-phosphate, a crucial compound in the synthesis of the core oligosaccharide. In a recent study, a HP0859 knockout mutant has been characterized, demonstrating a severe loss of lipopolysaccharide structure and a significant reduction of adhesion levels in an infection model to AGS cells, if compared with the wild type strain, in good agreement with its enzymatic role. The crystal structure reveals that the enzyme is a homo-pentamer, and NAD is bound as a cofactor in a highly conserved pocket. The substrate-binding site of the enzyme is very similar to that of its orthologue in Escherichia coli, suggesting also a similar catalytic mechanism.► LPS play an important role in maintaining the integrity of Gram-negative bacteria. ► HP0859 of Helicobacter pylori is a key enzyme in the biosynthesis of core LPS. ► Crystal structure of HP0859 reveals that the enzyme is a homo-pentamer. ► Each monomer presents two domains, a nucleotide- and a substrate- binding domain. ► NAD is bound as a cofactor.

Keywords: Abbreviations; r.m.s.d.; root mean square deviation; Hp; AGME; H. pylori; ADP-L-; glycero; -D-; manno; -heptose-6-epimerase; Ec; AGME; E. coli; ADP-L-; glycero; -D-; manno; -heptose-6-epimerase Helicobacter pylori; LPS; Lipopolysaccharide; SDR family

The crystal structure of ADP-L- glycero-D- manno-heptose-6-epimerase (HP0859) from Helicobacter pylori by Md Munan Shaik; Giuseppe Zanotti; Laura Cendron (pp. 1641-1647).

Opposing influences by subsite −1 and subsite +1 residues on relative xylopyranosidase/arabinofuranosidase activities of bifunctional β-D-xylosidase/α-L-arabinofuranosidase by Douglas B. Jordan; Jay D. Braker (pp. 1648-1657).

Conformational inversion occurs 7–8kcal/mol more readily in furanoses than pyranoses. This difference is exploited here to probe for active-site residues involved in distorting pyranosyl substrate toward reactivity. Spontaneous glycoside hydrolysis rates are ordered 4-nitrophenyl-α-l-arabinofuranoside (4NPA)>4-nitrophenyl-β-d-xylopyranoside (4NPX)>xylobiose ( X2). The bifunctional β-d-xylosidase/α-l-arabinofuranosidase exhibits the opposite order of reactivity, illustrating that the enzyme is well equipped in using pyranosyl groups of natural substrate X2 in facilitating glycoside hydrolysis. Probing the roles of all 17 active-site residues by single-site mutation to alanine and by changing both moieties of substrate demonstrates that the mutations of subsite −1 residues decrease the ratio k_cat^4NPX/4NPA, suggesting that the native residues support pyranosyl substrate distortion, whereas the mutations of subsite +1 and the subsite −1/+1 interface residues increase the ratio k_cat^4NPX/4NPA, suggesting that the native residues support other factors, such as C1 migration and protonation of the leaving group. Alanine mutations of subsite −1 residues raise k_cat^X2/4NPX and alanine mutations of subsite +1 and interface residues lower k_cat^X2/4NPX. We propose that pyranosyl substrate distortion is supported entirely by native residues of subsite −1. Other factors leading to the transition state are supported entirely by native residues of subsite +1 and interface residues.Display Omitted► Conformational inversion is 7–8kcal/mol greater in pyranoses than furanoses. ► Active-site residues of the two subsite enzyme were individually mutated to alanine. ► Mutants were probed with varied substrate occupants of subsite –1, +1, or both. ► We propose that residues involved in substrate distortion are in subsite –1. ► We propose that residues involved in other aspects of catalysis are in subsite +1.

Keywords: Abbreviations; SXA; β-; d; -xylosidase/α-L-arabinofuranosidase from; Selenomonas ruminantium; 4NPA; 4-nitrophenyl-α-; l; -arabinofuranoside; 4NPX; 4-nitrophenyl-β-; d; -xylopyranoside; X; 2; xylobiose; TEA; triethanolamine; ITC; isothermal titration calorimetry; GH; glycoside hydrolase; GH43; GH family 43; TS; transition stateSite-directed mutagenesis; Pyranosyl substrate distortion; Glycoside hydrolase; Plant cell wall deconstruction; Biomass saccharification

The mitochondrial proteome of the model legume Medicago truncatula by Juri Dubinin; Hans-Peter Braun; Udo Schmitz; Frank Colditz (pp. 1658-1668).

Legumes carry out special biochemical functions, e.g. the fixation of molecular nitrogen based on a symbiosis with proteobacteria. At the cellular level, this symbiosis has to be implemented into the energy metabolism of the host cell. To provide a basis for future analyses, we have characterized the protein complement of mitochondria of the model legume Medicago truncatula using two-dimensional isoelectric focussing (IEF) and blue-native (BN)-SDS-PAGE. While the IEF reference map resulted mainly in resolution of those proteins associated with the mitochondrial matrix, the BN proteomic map allowed separation of protein subunits from the respiratory chain protein complexes, which are located in the organelle's inner membrane. The M. truncatula mitochondrial BN reference map revealed some striking similarities to the one from Arabidopsis thaliana but at the same time exhibited also some special features: complex II is of increased abundance and additionally represented by a low molecular mass form not reported for Arabidopsis. Furthermore three highly abundant forms of prohibitin complexes are present in the mitochondrial proteome of M. truncatula. Special features with respect to mitochondrial protein complexes might reflect adaptations of legumes to elevated cellular energy requirements enabling them to develop symbiotic interactions with rhizobial bacteria.Display Omitted► The mitochondrial proteome of the model legume Medicago truncatula was analyzed. ► 195 proteins were identified in IEF and BN reference maps. ► Medicago reference maps revealed striking similarities to the one from Arabidopsis. ► Distinct features I: Complex II is increased in abundance and present in two forms. ► Distinct features II: three highly abundant prohibitin complexes are present.

Keywords: Medicago truncatula; Mitochondrial proteome; Blue-native (BN); SDS-PAGE; Complex II (succinate dehydrogenase); Mitochondrial prohibitin

The mitochondrial proteome of the model legume Medicago truncatula by Juri Dubinin; Hans-Peter Braun; Udo Schmitz; Frank Colditz (pp. 1658-1668).

Keywords: Medicago truncatula; Mitochondrial proteome; Blue-native (BN); SDS-PAGE; Complex II (succinate dehydrogenase); Mitochondrial prohibitin

Novel fungal phenylpyruvate reductase belongs tod-isomer-specific 2-hydroxyacid dehydrogenase family by Taiki Fujii; Motoyuki Shimizu; Yuki Doi; Tomoya Fujita; Takashi Ito; Daisuke Miura; Hiroyuki Wariishi; Naoki Takaya (pp. 1669-1676).

We discovered the phenyllactate (PLA)-producing fungal strain Wickerhamia fluorescens TK1 and purified phenylpyruvate reductase (PPR) from fungal cell-free extracts. The PPR used both NADPH and NADH as cofactors with more preference for the former. The enzyme reaction as well as the fungal culture produced optically actived-PLA. The gene for the PPR ( pprA) was cloned and expressed in Escherichia coli cells. Purified preparations of both native and recombinant PPR used hydroxyphenylpyruvate, glyoxylate and hydroxypyruvate as substrates but not pyruvate, oxaloacetate or benzoylformate. The predicted PPR protein had sequence similarity to proteins in thed-isomer-specific 2-hydroxyacid dehydrogenase family. Phylogenetic analyses indicated that the predicted PPR protein together with fungal predicted proteins constitutes a novel group of glyoxylate/hydroxypyruvate reductases. The fungus efficiently converted phenylalanine and phenylpyruvate tod-PLA. These compounds up-regulated the transcription of pprA, suggesting that it plays a role in fungal phenylalanine metabolism.► We discovered the phenyllactate-producing yeast Wickerhamia fluorescens TK1. ► Phenylpyruvate reductase was purified from the yeast's cell-free extracts. ► The phenylpyruvate reductase uses NADPH, phenylpyruvate, glyoxylate and hydroxypyruvate as substrates. ► Its encoding gene was cloned. ► The protein constitutes a novel group of glyoxylate/hydroxypyruvate reductases.

Keywords: Abbreviations; GHPR; glyoxylate/hydroxypyruvate reductase; HPLC; high-performance liquid chromatography; O.D.; optical density; PAT; phenylalanine aminotransferase; PLA; phenyllactic acid; PPA; phenylpyruvic acid; PPR; phenylpyruvate reductase; RACE; rapid amplification of cDNA ends; SDS–PAGE; sodium dodecylsulfate–polyacrylamide gel electrophoresis; SERA; d; -3-phosphoglycerate dehydrogenasePhenyllactate; Hydroxypyruvate; Glyoxylate reductase; Ascomycota; Wickerhamia fluorescens

Charged residues on a flap-loop structure of Lactococcus lactis prolidase play critical roles in allosteric behavior and substrate inhibition by Jian An Chen; Takuji Tanaka (pp. 1677-1685).

Allosteric behavior and substrate inhibition are unique characteristics of Lactococcus lactis prolidase. We hypothesized that charged residues (Asp36, His38, Glu39, and Arg40), present on one loop essential for catalysis, interact with residues in or near the active site to impart these unique characteristics. Asp36 has a predominant role in the allosteric behavior, as demonstrated through the non-allosteric behavior of the D36S mutant enzyme. In contrast, a double mutant (D36E/R293K) maintained the allostery, indicating that this aspartic acid residue interacts with Arg293, previously shown to be critical in the allostery. Substitution of His38 drastically reduced the substrate inhibition, and substrate specificity of the mutant at Asp36 or His38 showed the influence of these residues to the substrate specificity. These findings confirm the importance of the loop in the enzymatic reaction mechanism and suggest the existence of conformational changes of the loop structure between open and closed states. A variety of mutations at Glu39 and Arg40 showed that these residues influence roles of the loop in the enzyme reaction. On the basis of these results and combined with observations of molecular models of this prolidase, we concluded that Asp36 and His38 interact with the residues in the active site to generate an allosteric subsite and a pseudo- S₁′ site, which are responsible for the allosteric behavior and substrate inhibition.► Charged resides on an active site loop are critical to exhibit the activity. ► Asp36 interacts with Arg293, resulting in allosteric behavior. ► His38 forms a pseudo-S1′ site that causes the substrate inhibition.

Keywords: Abbreviations; Lla; prol; Lactococcus lactis; prolidase; Pf; prol; Pyrococcus furiosus; prolidaseProline specific peptidase; Structure–function relationship; Allosteric subsite; Pseudo substrate subsite; Intersubunit interaction

Charged residues on a flap-loop structure of Lactococcus lactis prolidase play critical roles in allosteric behavior and substrate inhibition by Jian An Chen; Takuji Tanaka (pp. 1677-1685).

Kinetic mechanism of an aldehyde reductase of Saccharomyces cerevisiae that relieves toxicity of furfural and 5-hydroxymethylfurfural by Douglas B. Jordan; Jay D. Braker; Michael J. Bowman; Karl E. Vermillion; Jaewoong Moon; Z. Lewis Liu (pp. 1686-1694).

An effective means of relieving the toxicity of furan aldehydes, furfural (FFA) and 5-hydroxymethylfurfural (HMF), on fermenting organisms is essential for achieving efficient fermentation of lignocellulosic biomass to ethanol and other products. Ari1p, an aldehyde reductase from Saccharomyces cerevisiae, has been shown to mitigate the toxicity of FFA and HMF by catalyzing the NADPH-dependent conversion to corresponding alcohols, furfuryl alcohol (FFOH) and 5-hydroxymethylfurfuryl alcohol (HMFOH). At pH 7.0 and 25°C, purified Ari1p catalyzes the NADPH-dependent reduction of substrates with the following values ( k_cat (s−¹), k_cat/ K_m (s−¹mM−¹), K_m (mM)): FFA (23.3, 1.82, 12.8), HMF (4.08, 0.173, 23.6), anddl-glyceraldehyde (2.40, 0.0650, 37.0). When acting on HMF anddl-glyceraldehyde, the enzyme operates through an equilibrium ordered kinetic mechanism. In the physiological direction of the reaction, NADPH binds first and NADP⁺ dissociates from the enzyme last, demonstrated by k_cat of HMF anddl-glyceraldehyde that are independent of [NADPH] and ( K_ia^NADPH/ k_cat) that extrapolate to zero at saturating HMF ordl-glyceraldehyde concentration. Microscopic kinetic parameters were determined for the HMF reaction (HMF+NADPH↔HMFOH+NADP⁺), by applying steady-state, presteady-state, kinetic isotope effects, and dynamic modeling methods. Release of products, HMFOH and NADP⁺, is 84% rate limiting to k_cat in the forward direction. Equilibrium constants, [NADP⁺][FFOH]/[NADPH][FFA][H⁺]=5600×10⁷M−¹ and [NADP⁺][HMFOH]/[NADPH][HMF][H⁺]=4200×10⁷M−¹, favor the physiological direction mirrored by the slowness of hydride transfer in the non-physiological direction, NADP⁺-dependent oxidation of alcohols ( k_cat (s−¹), k_cat/ K_m (s−¹mM−¹), K_m (mM)): FFOH (0.221, 0.00158, 140) and HMFOH (0.0105, 0.000104, 101).► Ari1p reduces furan aldehydes to their less toxic alcohols in yeast. ► At equilibrium, reactions favor the alcohol and NADP⁺. ► K_eq [NADP⁺][furfuryl alcohol]/[NADPH][furfural][H⁺]=5600x10⁷ M^–1. ► Catalyzed reactions are equilibrium ordered: NADPH binds first and NADP⁺ leaves last. ► Microscopic rate constants indicate release of products is 84% rate-limiting to k_cat.

Keywords: Abbreviations; FFA; furfural; HMF; hydroxymethylfurfural; FFOH; furfuryl alcohol; HMFOH; 5-hydroxymethylfurfuryl alcohol; Ari1p; an aldehyde reductase from; Saccharomyces cerevisiae; KIE; kinetic isotope effectFurfural reductase; Short chain dehydrogenase/reductase; Bioenergy; Detoxification; Fermentation

A thermostable esterase from Thermoanaerobacter tengcongensis opening up a new family of bacterial lipolytic enzymes by Lang Rao; Yanfen Xue; Cheng Zhou; Jin Tao; Gang Li; Jian R. Lu; Yanhe Ma (pp. 1695-1702).

An unidentified α/β hydrolase gene lipA3 from thermostable eubacterium species Thermoanaerobacter tengcongensis MB4 was cloned and heterologously expressed by Escherichia coli BL21(DE3)pLysS. The purified recombinant enzyme EstA3 turned out to be a monomeric thermostable esterase with optimal activity at 70°C and pH 9.5. The enzyme showed lipolytic activity towards a wide range of ester substrates including p-nitrophenyl esters and triacylglycerides, with the highest activity being observed for p-nitrophenyl caproate at 150 U/mg and for Triacetin at 126U/mg, respectively. Phylogenetic analysis revealed that EstA3 did not show homology to any identified bacterial lipolytic hydrolases. Sequence alignment showed that there was a common pentapeptide CHSMG with a cysteine replacing the first glycine in most esterase and lipase conserved motif GXSXG. The catalytic triad of EstA3 is Ser92, Asp269 and His292, which was confirmed by site directed mutagenesis. Based on the enzymatic properties and sequence alignment we concluded that the esterase EstA3 represented a novel bacterial lipolytic enzyme group and in chorological order this group was assigned as Family XIV.► A new thermophilic enzyme called LipA3 has been expressed and purified. ► It falls into α/β hydrolase type. ► It is a monomeric thermostable esterase with optimal activity at 70°C and pH 9.5. ► On the basis of enzymatic properties and sequence alignment we concluded that it is a new group of bacterial lipolytic enzymes. ► The group was assigned as Family XIV in chorological order.

Keywords: Thermophile; Esterase; Lipolytic family; α/β hydrolase superfamily; Enzyme; Extremophile

Conformation-dependent scFv antibodies specifically recognize the oligomers assembled from various amyloids and show colocalization of amyloid fibrils with oligomers in patients with amyloidoses by Xi Zhang; Xiao-xia Sun; Di Xue; Dong-ge Liu; Xiao-yan Hu; Min Zhao; Shi-gao Yang; Yang Yang; Yong-jing Xia; Ying Wang; Rui-tian Liu (pp. 1703-1712).

Increasing evidence indicates that amyloid aggregates, including oligomers, protofibrils or fibrils, are pivotal toxins in the pathogenesis of many amyloidoses such as Alzheimer's disease (AD), Parkinson's disease, Huntington's disease, prion-related diseases, type 2 diabetes and hereditary renal amyloidosis. Various oligomers assembled from different amyloid proteins share common structures and epitopes. Here we present data indicating that two oligomer-specific single chain variable fragment (scFv) antibodies isolated from a naïve human scFv library could conformation-dependently recognize oligomers assembled from α-synuclein, amylin, insulin, Aβ1–40, prion peptide 106–126 and lysozyme, and fibrils from lysozyme. Further investigation showed that both scFvs inhibited the fibrillization of α-synuclein, amylin, insulin, Aβ1–40 and prion peptide 106–126, and disaggregated their preformed fibrils. However, they both promoted the aggregation of lysozyme. Nevertheless, the two scFv antibodies could attenuate the cytotoxicity of all amyloids tested. Moreover, the scFvs recognized the amyloid oligomers in all types of plaques, Lewy bodies and amylin deposits in the brain tissues of AD and PD patients and the pancreas of type 2 diabetes patients respectively, and showed that most amyloid fibril deposits were colocalized with oligomers in the tissues. Such conformation-dependent scFv antibodies may have potential application in the investigation of aggregate structures, the mechanisms of aggregation and cytotoxicity of various amyloids, and in the development of diagnostic and therapeutic reagents for many amyloidoses.► The oligomer-specific scFv antibodies recognize oligomers of various amyloid. ► The scFv inhibited fibrillization and cytotoxicity of various amyloids. ► The scFvs disaggregated preformed amyloid fibrils. ► The scFvs recognized the amyloid oligomers in the tissues of patients. ► Most amyloid fibril deposits were colocalized with oligomers in the tissues.

Keywords: Abbreviations; AD; Alzheimer's disease; PD; Parkinson's disease; Aβ; beta-amyloid peptide; PrP; prion protein; scFv; single chain variable fragment; ThT; thioflavin T; ThS; thioflavin S; TEM; Transmission Electron Microscope; MTT; 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromideAmyloidosis; Amyloid; Single-chain variable fragment antibody; Fibril deposit

Molecular characterization of endo-1,3-β-glucanase from Cellulosimicrobium cellulans: Effects of carbohydrate-binding module on enzymatic function and stability by Yoichi Tanabe; Masayuki Oda (pp. 1713-1719).

An endo-1,3-β-glucanase was purified from Tunicase®, a crude enzyme preparation from Cellulosimicrobium cellulans DK-1, and determined to be a 383-residue protein (Ala1-Leu383), comprising a catalytic domain of the glycoside hydrolase family 16 and a C-terminal carbohydrate-binding module family 13. The Escherichia coli expression system of the catalytic domain (Ala1-Thr256) was constructed, and the protein with N-terminal polyhistidine tag was purified using a Ni-nitrilotriacetic acid column. We analyzed enzymatic properties of the recombinant catalytic domain, its variants, and the Tunicase®-derived full-length endo-1,3-β-glucanase. Substitution of Glu119 with Ala and deletion of Met123, both of the residues are located in the catalytic motif, resulted in the loss of hydrolytic activity. In comparison between the full-length enzyme and isolated catalytic domain, their hydrolytic activities for soluble substrates such as laminarin and laminarioligosaccharides were similar. In contrast, the hydrolytic activity of the full-length enzyme for insoluble substrates such as curdlan and yeast-glucan was significantly higher than that of the catalytic domain. It should be noted that the acid stabilities for the hydrolysis of laminarin were clearly different. Secondary structure analysis using circular dichroism showed that the full-length enzyme was more acid stable than was the catalytic domain, possibly because of domain interactions between the catalytic domain and the carbohydrate-binding module.► The endo-1,3-b-glucanase comprises a catalytic domain and CBM13. ► Glu119 and Met123 are critically important residues for the catalysis. ► The CBM13 has little effects on the hydrolysis of soluble substrates. ► The CBM13 contributes to the hydrolysis of insoluble substrates and acid tolerance.

Keywords: Abbreviations; GH16; glycoside hydrolase family 16; CBM13; carbohydrate-binding module family 13; E. coli; Escherichia coli; CMC; carboxymethyl cellulose; PCR; polymerase chain reaction; NTA; Ni-nitrilotriacetic acid; His-tag; polyhistidine-tag; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DEAE; diethyl amino ethyl; MALDI-TOF MS; matrix assisted laser desorption/ionization-time of flight mass spectrometry; CD; circular dichroismCatalytic domain; Carbohydrate-binding module; Enzymatic activity; β-1,3 glucan; Acid tolerance

Recombinant production and characterisation of two related GH5 endo-β-1,4-mannanases from Aspergillus nidulans FGSC A4 showing distinctly different transglycosylation capacity by Adiphol Dilokpimol; Hiroyuki Nakai; Charlotte H. Gotfredsen; Martin J. Baumann; Natsuko Nakai; Maher Abou Hachem; Birte Svensson (pp. 1720-1729).

The glycoside hydrolase family 5 (GH5) endo-β-1,4-mannanases ManA and ManC from Aspergillus nidulans FGSC A4 were produced in Pichia pastoris X33 and purified in high yields of 120 and 145mg/L, respectively, from the culture supernatants. Both enzymes showed increasing catalytic efficiency ( k_cat/ K_M) towards β-1,4 manno-oligosaccharides with the degree of polymerisation (DP) from 4 to 6 and also hydrolysed konjac glucomannan, guar gum and locust bean gum galactomannans. ManC had up to two-fold higher catalytic efficiency for DP 5 and 6 manno-oligosaccharides and also higher activity than ManA towards mannans. Remarkably, ManC compared to ManA transglycosylated mannotetraose with formation of longer β-1,4 manno-oligosaccharides 8-fold more efficiently and was able to use mannotriose, melezitose and isomaltotriose out of 36 tested acceptors resulting in novel penta- and hexasaccharides, whereas ManA used only mannotriose as acceptor. ManA and ManC share 39% sequence identity and homology modelling suggesting that they have very similar substrate interactions at subsites +1 and +2 except that ManC Trp283 at subsite +1 corresponded to Ser289 in ManA. Site-directed mutagenesis to ManA S289W lowered K_M for manno-oligosaccharides by 30–45% and increased transglycosylation yield by 50% compared to wild-type. Conversely, K_M for ManC W283S was increased, the transglycosylation yield was reduced by 30–45% and furthermore activity towards mannans decreased below that of ManA. This first mutational analysis in subsite +1 of GH5 endo-β-1,4-mannanases indicated that Trp283 in ManC participates in discriminating between mannan substrates with different extent of branching and has a role in transglycosylation and substrate affinity.► First report of hetero-mannooligosaccharides production by transmannosylation. ► Differential transglycosylation by two recombinant GH5 mannanases. ► Novel penta- and hexasaccharides synthesised by ManC. ► First mutational analysis of subsite +1 in GH5 mannanases. ► ManC Trp283 is a transglycosylation and mannan-type determinant.

Keywords: Abbreviations; DP; degree of polymerisation; GH; glycoside hydrolase family; Hj; Man5A; endo-β-1,4-mannanase from; Hypocrea jecorina; RUTC-30 (GenBank AAA34208.1); HPAEC-PAD; high performance anion exchange chromatography with pulsed amperometric detection; IMAC; immobilised metal ion affinity chromatography; M1; mannose; M2; mannobiose; M3; mannotriose; M4; mannotetraose; M5; mannopentaose; M6; mannohexaose; M7; mannoheptaose; ManA and ManC; GH5 endo-β-1,4-mannanase from; Aspergillus nidulans; FGSC A4 (GenBank EAA63326.1 and EAA58449.1, respectively); Tf; Man; GH5 endo-β-1,4-mannanase from; Thermobifida fusca; KW3 (GenBank CAA06924.1)Endo-β-1,4-mannanase; Transglycosylation; Hetero-mannooligosaccharide; Mannan; Subsite +; 1 mutagenesis

Chaperonins induce an amyloid-like transformation of ovine prion protein: The fundamental difference in action between eukaryotic TRiC and bacterial GroEL by Georgy G. Kiselev; Irina N. Naletova; Evgeny V. Sheval; Yulia Y. Stroylova; Elena V. Schmalhausen; Haertle Thomas Haertlé; Vladimir I. Muronetz (pp. 1730-1738).

Molecular chaperones have been shown to be involved in the processes taking place during the pathogenesis of various amyloid neurodegenerative diseases. However, contradictory literature reports suggest that different molecular chaperones can either stimulate or prevent the formation of amyloid structures from distinct amyloidogenic proteins. In the present work, we concentrated on the effects caused by two molecular chaperonins, ovine TRiC and bacterial GroEL, on the aggregation and conformational state of ovine PrP. Both chaperonins were shown to bind native PrP and to produce amyloid-like forms of ovine PrP enriched with beta-structures but, while GroEL acted in an ATP-dependent manner, TRiC was shown to cause the same effect only in the absence of Mg-ATP ( i. e. in the inactive form). In the presence of chaperonin GroEL, ovine PrP was shown to form micellar particles, approximately 100–200nm in diameter, which were observed both by dynamic light scattering assay and by electron microscopy. The content of these particles was significantly higher in the presence of Mg-ATP and, only under these conditions, GroEL produced amyloid-like species enriched with beta-structures. TRiC was shown to induce the formation of amyloid fibrils observed by electron microscopy, but only in the absence of Mg-ATP. This study suggests the important role of the cytosolic chaperonin TRiC in the propagation of amyloid structures in vivo during the development of amyloid diseases and the possible role of the bacterial chaperonin GroEL, located in the intestinal microflora, in the induction of these diseases.Display Omitted► We compared effects caused by bacterial GroEL and eukaryotic TRiC on the ovine PrP. ► Both chaperonins bind the native prion protein. ► GroEL produces amyloid-like forms of ovine PrP in the presence of Mg-ATP. ► TRiC produces amyloid-like forms of ovine PrP only in the absence of Mg-ATP. ► Chaperonin TRiC induces the formation of amyloid fibrils from ovine PrP.

Keywords: Abbreviations; GroEL; Growth factor E, Large subunit (bacterial chaperonin); GroES; Growth factor E, Small subunit (bacterial co-chaperonin); TRiC; TCP-1 Ring Complex - eukaryotic cytosolic chaperonin (also known as CCT, Chaperonin Containing TSP-1); Hsp; Heat shock protein(s); ovPrP-VRQ; allelic variant of ovine prion protein (V136, R154, Q171, respectively); PrP; prion protein of vertebrates; ThT; Thioflavin TMammalian prion protein; Chaperonin GroEL; Chaperonin TRiC; Amyloid-like transformation; Amyloid fibril formation; Intestinal microflora

Insight into the binding of the wild type and mutated alginate lyase (AlyVI) with its substrate: A computational and experimental study by Adel Hamza; Yu Lan Piao; Mi-Sun Kim; Cheol Hee Choi; Chang-Guo Zhan; Hoon Cho (pp. 1739-1747).

The homology model of the wild type alginate lyase (AlyVI) marine bacterium Vibrio sp. protein, was built using the crystal structure of the Family 7 alginate lyase from Sphingomonas sp. A1. To rationalize the observed structure–affinity relationships of aliginate lyase alyVI with its (GGG) substrate, molecular docking, MD imulations and binding free energy calculations followed by site-directed mutagenesis and alyVI activity assays were carried out. Per-residue decomposition of the (GGG) binding energy revealed that the most important contributions were from polar and charged residues, such as Asn138, Arg143, Asn217, and Lys308, while van der Waals interactions were responsible for binding with the catalytic His200 and Tyr312 residues. The mutants H200A, K308A, Y312A, Y312F, and W165A were found to be inactive or almost inactive. However, the catalytic efficiency ( k_cat/ K_m) of the double mutant L224V/D226G increased by two-fold compared to the wild type enzyme. This first structural model with its substrate binding mode and the agreement with experimental results provide a suitable base for the future rational design of new mutated alyVI structures with improved catalytic activity.

Keywords: Alginate lyase; AlyVI; Homology modeling; Docking; Site-directed mutagenesis; MD simulations

Elongin C is a mediator of Notch4 activity in human renal tubule cells by Timothy D. Cummins; Michael D. Mendenhall; Michelle N. Lowry; Erik A.Korte; Michelle T. Barati; Syed J. Khundmiri; Sarah A. Salyer; Jon B. Klein; David W. Powell (pp. 1748-1757).

Notch proteins (Notch 1–4) are a family of trans-membrane cell surface receptors that are converted into transcriptional regulators when activated by interactions with cell surface ligands on adjacent cells. Ligand-binding stimulates proteolytic cleavage of the trans-membrane domain, releasing an active intracellular domain (ICD) that translocates to the nucleus and impacts transcription. In transit, the ICD may interact with regulatory proteins that modulate the expression and transcriptional activity. We have found that Notch4^ICD expression is enhanced in the tubule cells of fibrotic kidneys from diabetic mice and humans and identified Notch4^ICD interacting proteins that could be pertinent to normal and pathological functions. Using proteomic techniques, several components of the Elongin C complex were identified as candidate Notch4^ICD interactors. Elongin C complexes can function as ubiquitin ligases capable of regulating proteasomal degradation of specific protein substrates. Our studies indicate that ectopic Elongin C expression stimulates Notch4^ICD degradation and inhibits its transcriptional activity in human kidney tubule HK11 cells. Blocking Elongin C mediated degradation by MG132 indicates the potential for ubiquitin-mediated Elongin C regulation of Notch4^ICD. Functional interaction of Notch4^ICD and Elongin C provides novel insight into regulation of Notch signaling in epithelial cell biology and disease.► Notch4^ICD acts in concert with TGF-β pathway to drive fibrotic signaling. ► Proteomics studies indicate Notch4^ICD and elongin C interact. ► Functional studies show elongin C inhibits Notch4-mediated TGF-β fibrotic activity. ► Elongin C negatively regulates Notch4^ICD in a proteasome dependent manner. ► Elongin C is minimally expressed in type 1 diabetic renal proximal tubule cells.

Keywords: Notch signaling; TGF-beta; Proteasomal degradation; Ubiquitin ligase; Proteomics; Mass spectrometry

Characterization of JBURE-IIb isoform of Canavalia ensiformis (L.) DC urease by Fernanda Mulinari; Arlete Beatriz Becker-Ritt; Diogo Ribeiro Demartini; Rodrigo Ligabue-Braun; Staniscuaski Fernanda Stanisçuaski; Hugo Verli; Rodrigo R. Fragoso; Evelyn Koeche Schroeder; Célia Regina Carlini; Grossi-de-Sa Maria Fátima Grossi-de-Sá (pp. 1758-1768).

Ureases, nickel-dependent enzymes that catalyze the hydrolysis of urea into ammonia and bicarbonate, are widespread in plants, bacteria, and fungi. Previously, we cloned a cDNA encoding a Canavalia ensiformis urease isoform named JBURE-II, corresponding to a putative smaller urease protein (78kDa) when compared to other plant ureases. Aiming to produce the recombinant protein, we obtained jbure- IIb, with different 3′ and 5′ ends, encoding a 90kDa urease. Three peptides unique to the JBURE-II/-IIb protein were detected by mass spectrometry in seed extracts, indicating that jbure- II/- IIb is a functional gene. Comparative modeling indicates that JBURE-IIb urease has an overall shape almost identical to C. ensiformis major urease JBURE-I with all residues critical for urease activity. The cDNA was cloned into the pET101 vector and the recombinant protein was produced in Escherichia coli. The JBURE-IIb protein, although enzymatically inactive presumably due to the absence of Ni atoms in its active site, impaired the growth of a phytopathogenic fungus and showed entomotoxic properties, inhibiting diuresis of Rhodnius prolixus isolated Malpighian tubules, in concentrations similar to those reported for JBURE-I and canatoxin. The antifungal and entomotoxic properties of the recombinant JBURE-IIb apourease are consistent with a protective role of ureases in plants.► A cDNA encoding the isoform JBURE-IIb of Canavalia ensiformis urease was cloned. ► Comparative modeling and phylogenetic analysis of JBURE-IIb were performed. ► Proteomic analysis of C. ensiformis indicated that jbure- IIb is a functional gene. ► The full length jbure- IIb cDNA was cloned and expressed in Escherichia coli. ► Biological properties of JBURE-IIb point to a protective role of ureases in plants.

Keywords: Abbreviations; ABA; abscisic acid; CNTX; canatoxin; DTT; dithiothreitol; JBURE-I; jack bean major urease; JBURE-II; jack bean urease isoform II; RACE; rapid amplification of cDNA ends; RT-PCR; reverse transcription and polymerase chain reactionAntifungal protein; Comparative modeling; Heterologous expression; Insecticide; Jackbean urease; Ni metallocenter

The effect of deleting residue C269 in the β12–β13 loop of protein phosphatase 2A (PP2A)catalytic subunit on the interaction between PP2A and metal ions, especially Mn²⁺ by Hui Li; Chao Liu; Hao Zhang; Qun Wei (pp. 1769-1774).

Protein phosphatase 2A (PP2A) is one of the most important Ser/Thr phosphatases in eukaryotic cells. The enzymatic core of PP2A (PP2A_D) consists of a scaffold subunit (A subunit) and a catalytic subunit (C subunit). When residue Cys269 in the β12–β13 loop of the PP2A C subunit was deleted (ΔC269), the activity and the intrinsic fluorescence intensity of PP2A_D decreased. Specify the effects of some metal ions on PP2A_D were also changed. Mn²⁺ in particular was an efficient activator of ΔC269 and altered the intrinsic fluorescence spectrum of ΔC269. Remarkably, after pre-treatment of ΔC269 with Mn²⁺, the effects of other metal ions showed the same trends as they had on the WT. Molecular dynamics (MD) simulations showed that deletion of Cys269 decreased the polarity of the β12–β13 loop of PP2A Cα. We conclude that deletion of residue Cys269 alters the conformation and activity of PP2A_D and influences the interaction between PP2A and various metal ions, notably Mn²⁺.► The deletion of Cys269 in PP2AC influences the activity of the PP2AD and changes the 3D structure of PP2AC. ► Cys269 deletion affects the interaction between PP2A and metal ions, especially Mn²⁺. ► C269 adjusts the polarity of the β12–β13 loop of PP2AC.

Keywords: C269; PP2A; Metal ion; Mutant; Mn; ²⁺

Solubilization and folding of a fully active recombinant Gaussia luciferase with native disulfide bonds by using a SEP-Tag by Tharangani Rathnayaka; Minako Tawa; Takashi Nakamura; Shihori Sohya; Kunihiro Kuwajima; Masafumi Yohda; Yutaka Kuroda (pp. 1775-1778).

Gaussia luciferase (GLuc) is the smallest known bioluminescent protein and is attracting much attention as a potential reporter protein. However, its 10 disulfide bond forming cysteines have hampered the efficient production of recombinant GLuc and thus limited its use in bio-imaging application. Here, we demonstrate that the addition of a short solubility enhancement peptide tag (SEP-Tag) to the C-terminus of GLuc (GLuc-C9D) significantly increased the fraction of soluble protein at a standard expression temperature. The expression time was much shorter, and the final yield of GLuc-C9D was significantly higher than with our previous pCold expression system. Reversed phase HPLC indicated that the GLuc-C9D variant folded with a single disulfide bond pattern after proper oxidization. Further, the thermal denaturation of GLuc-C9D was completely reversible, and its secondary structure content remained unchanged until 40°C as assessed by CD spectroscopy. The¹H-NMR spectrum of GLuc indicated sharp well dispersed peaks typical for natively folded proteins. GLuc-C9D bioluminescence activity was strong and fully retained even after incubation at high temperatures. These results suggest that solubilization using SEP-Tags can be useful for producing large quantities of proteins containing multiple disulfide bonds.► Efficient production of recombinant GLuc is hampered by its 10 cysteines. ► Fusion of a SEP-Tag significantly increased the production of soluble GLuc in E. coli. ► The solubilized GLuc was fully functional with a native-like disulfide bond pattern.

Keywords: Abbreviations; GLuc; Gaussia; Luciferase; SEP-Tag; Solubility Enhancement Peptide Tag; pCold-GLuc; wild type GLuc cloned in pCold vector; GLuc-C9D; wild type GLuc cloned in pAED4 vector flanked with a N terminus His tag and a C terminus nine aspartic acid tag; GLuc-C0D; wild type GLuc sequence cloned in pAED4 vector with a N terminus His tagBioluminescence; Protein folding; Protein solubility; Aggregation; Disulfide bond

Contacts between mammalian mitochondrial translational initiation factor 3 and ribosomal proteins in the small subunit by Md. Emdadul Haque; Hasan Koc; Huseyin Cimen; Emine C. Koc; Linda L. Spremulli (pp. 1779-1784).

Mammalian mitochondrial translational initiation factor 3 (IF3_mt) binds to the small subunit of the ribosome displacing the large subunit during the initiation of protein biosynthesis. About half of the proteins in mitochondrial ribosomes have homologs in bacteria while the remainder are unique to the mitochondrion. To obtain information on the ribosomal proteins located near the IF3_mt binding site, cross-linking studies were carried out followed by identification of the cross-linked proteins by mass spectrometry. IF3_mt cross-links to mammalian mitochondrial homologs of the bacterial ribosomal proteins S5, S9, S10, and S18-2 and to unique mitochondrial ribosomal proteins MRPS29, MRPS32, MRPS36 and PTCD3 (Pet309) which has now been identified as a small subunit ribosomal protein. IF3_mt has extensions on both the N- and C-termini compared to the bacterial factors. Cross-linking of a truncated derivative lacking these extensions gives the same hits as the full length IF3_mt except that no cross-links were observed to MRPS36. IF3 consists of two domains separated by a flexible linker. Cross-linking of the isolated N- and C-domains was observed to a range of ribosomal proteins particularly with the C-domain carrying the linker which showed significant cross-linking to several ribosomal proteins not found in prokaryotes.► Mitochondria synthesize 13 proteins using a specialized protein biosynthetic system. ► Mitochondrial ribosomes have many proteins with no homologs in other systems. ► Ribosomal proteins at the binding site of initiation factor 3 were identified. ► IF3_mt has very different contacts with the ribosome than observed with bacterial IF3.

Keywords: Initiation factor 3; Mitochondrion; Ribosome; Ribosomal protein; Protein synthesis; Mammal

Circulating biomarkers of protein oxidation for Alzheimer disease: Expectations within limits by Fabio Di Domenico; Raffaella Coccia; D. Allan Butterfield; Marzia Perluigi (pp. 1785-1795).

Alzheimer disease (AD), the most common dementing disorder, is a multifactorial disease with complex etiology. Among different hypotheses proposed for AD one of the most corroborated is the “oxidative stress hypothesis”. Although recent studies extensively demonstrated the specific oxidative modification of selected proteins in the brain of AD patients and how their dysfunction possibly correlates with the pathology, there is still an urgent need to extend these findings to peripheral tissue. So far very few studies showed oxidative damage of proteins in peripheral tissues and current findings need to be replicated. Another limit in AD research is represented by the lack of highly specific diagnostic tools for early diagnosis. For a full screening and early diagnosis, biomarkers easily detectable in biological samples, such as blood, are needed. The search of reliable biomarkers for AD in peripheral blood is a great challenge. A few studies described a set of plasma markers that differentiated AD from controls and were shown to be useful in predicting conversion from mild cognitive impairment, which is considered a prodromal stage, to AD. We review the current state of knowledge on peripheral oxidative biomarkers for AD, including proteomics, which might be useful for early diagnosis and prognosis.

Keywords: Protein oxidation; Alzheimer disease; Biomarker; Body fluid; Redox proteomics

Circulating biomarkers of protein oxidation for Alzheimer disease: Expectations within limits by Fabio Di Domenico; Raffaella Coccia; D. Allan Butterfield; Marzia Perluigi (pp. 1785-1795).

Keywords: Protein oxidation; Alzheimer disease; Biomarker; Body fluid; Redox proteomics

Role of pH on dimeric interactions for DENV envelope protein: An insight from molecular dynamics study by Kshatresh Dutta Dubey; Amit Kumar Chaubey; Rajendra Prasad Ojha (pp. 1796-1801).

The entry of dengue viruses is mediated by pH triggering in the host cells. Here we have studied the DENV E protein stability and binding of its units at low and normal pH using MD and MM-PB/SA method for the first time. To investigate the role of pH in dissociation of dimeric protein, we have performed a concise study of hydrogen bonding and other interactions between units of dimer at low and normal pH. The Generalized Born calculation connotes that dimeric unit was relatively less stable and less proned for dimerisation at low pH. Our results provide a theoretical verification for previous assumptions of pH triggering mechanism of dengue envelope protein. During the pH alteration, we found a large decrement in salt bridges which were observed at normal pH. We also compared the flexibility of each unit and found that they exhibit different fluctuations during molecular dynamics simulations.► We performed MD study at normal and low pH. ► We calculated hydrogen bonding in normal and low pH. ► We have calculated interaction between dimer of DENV envelope protein. ► We investigated the inter-chain salt-bridges and also calculated thermal fluctuation. ► The stability and binding energy of complex are calculated at normal and low pH.

Keywords: Envelope protein; MM-PB (GB)/SA; Hydrogen bonding; Salt-bridge; pH variation

Role of pH on dimeric interactions for DENV envelope protein: An insight from molecular dynamics study by Kshatresh Dutta Dubey; Amit Kumar Chaubey; Rajendra Prasad Ojha (pp. 1796-1801).

Keywords: Envelope protein; MM-PB (GB)/SA; Hydrogen bonding; Salt-bridge; pH variation

Mycobacterium leprae RecA is structurally analogous but functionally distinct from Mycobacterium tuberculosis RecA protein by K. Neelakanteshwar Patil; Pawan Singh; Sri Harsha; K. Muniyappa (pp. 1802-1811).

Mycobacterium leprae is closely related to Mycobacterium tuberculosis, yet causes a very different illness. Detailed genomic comparison between these two species of mycobacteria reveals that the decaying M. leprae genome contains less than half of the M. tuberculosis functional genes. The reduction of genome size and accumulation of pseudogenes in the M. leprae genome is thought to result from multiple recombination events between related repetitive sequences, which provided the impetus to investigate the recombination-like activities of RecA protein. In this study, we have cloned, over-expressed and purified M. leprae RecA and compared its activities with that of M. tuberculosis RecA. Both proteins, despite being 91% identical at the amino acid level, exhibit strikingly different binding profiles for single-stranded DNA with varying GC contents, in the ability to catalyze the formation of D-loops and to promote DNA strand exchange. The kinetics and the extent of single-stranded DNA-dependent ATPase and coprotease activities were nearly equivalent between these two recombinases. However, the degree of inhibition exerted by a range of ATP:ADP ratios was greater on strand exchange promoted by M. leprae RecA compared to its M. tuberculosis counterpart. Taken together, our results provide insights into the mechanistic aspects of homologous recombination and coprotease activity promoted by M. lepare RecA, and further suggests that it differs from the M. tuberculosis counterpart. These results are consistent with an emerging concept of DNA-sequence influenced structural differences in RecA nucleoprotein filaments and how these differences reflect on the multiple activities associated with RecA protein.► M. leprae RecA protein (MlRecA) is purified to homogeneity and characterized. ► Functional studies indicate that MlRecA is different from M. tuberculosis RecA. ► M. leprae RecA promoted strand exchange is inversely proportional to the GC content. ► These results suggest DNA-sequence dependent differences in RecA-ssDNA filaments.

Keywords: Abbreviations; bp; base pair; BPB; bromophenol blue; dsDNA; double-stranded DNA; DTT; dithiothreitol; EcRecA; Escherichia coli; RecA; EDTA; ethylene diamine tetraacetic acid; EMSA; electrophoretic mobility shift assay; HR; homologous recombination; IPTG; isopropyl-1-thio-β-; d; galactopyranoside; MtRecA; Mycobacterium tuberculosis; RecA; MtLexA; Mycobacterium tuberculosis; LexA; MlRecA; Mycobacterium leprae; RecA; ODN; oligonucleotide; PAGE; polyacrylamide gel electrophoresis; SDS; sodium dodecyl sulfate; SE; strand exchange; ssDNA; single-stranded DNAMycobacterium; RecA; Gene decay; DNA strand exchange; ATPase; SOS response

A fraction of the transcription factor TAF15 participates in interactions with a subset of the spliceosomal U1 snRNP complex by Michael Leichter; Marija Marko; Vassiliki Ganou; Meropi Patrinou-Georgoula; László Tora; Apostolia Guialis (pp. 1812-1824).

RNA/ssDNA-binding proteins comprise an emerging class of multifunctional proteins with an anticipated role in coupling transcription with RNA processing. We focused here on the highly related transcription factors of the TET sub-class: TLS/FUS, EWS and in particular the least studied member TAF15. An extensive array of immunoprecipitation studies on differentially extracted HeLa nuclei revealed the specific association of TAF15 with the spliceosomal U1 snRNP complex, as deduced by the co-precipitating U1 snRNA, U1-70K and Sm proteins. Additionally, application of anti-U1 RNP autoantibodies identified TAF15 in the immunoprecipitates. Minor fractions of nuclear TAF15 and U1 snRNP were involved in this association. Pull-down assays using recombinant TAF15 and U1 snRNP-specific proteins (U1-70K, U1A and U1C) provided in vitro evidence for a direct protein–protein interaction between TAF15 and U1C, which required the N-terminal domain of TAF15. The ability of TAF15 to directly contact RNA, most likely RNA pol II transcripts, was supported by in vivo UV cross-linking studies in the presence of α-amanitin. By all findings, the existence of a functionally discrete subset of U1 snRNP in association with TAF15 was suggested and provided further support for the involvement of U1 snRNP components in early steps of coordinated gene expression.► The transcription factor TAF15 can associate with the spliceosomal U1 snRNP complex. ► TAF15 can make direct interactions with the U1C protein, in vitro. ► TAF15 can directly contact RNA, in vivo.

Keywords: Abbreviations; snRNA; small nuclear RNA; snRNP; small nuclear ribonucleoprotein; hnRNP; heterogeneous nuclear ribonucleoprotein; TBP; TATA-binding protein; TAF; TBP-associated factor; CTD; carboxy-terminal domain; RNA pol II; RNA polymerase II; SR; serine/arginine-rich; TFIID; transcription factor IID; EWS; Ewing's sarcoma; TLS/FUS; translocated in liposarcoma/fusion; TET; TLS/FUS, EWS and TAF15 proteins; RRM; RNA recognition motif; RBD; RNA binding domain; RGG; Arg-Gly-Gly motif; ssDNA; single strand DNA; GST; glutathione S-transferase; IP; immunoprecipitation; TIA-1; T-cell internal antigen-1; 2-D; two dimensional; NEPHGE; non-equilibrium pH gradient electrophoresisTET protein; Spliceosomal snRNP; RNA pol II transcription; RNA splicing; RNA/ssDNA-binding protein; TAF15-U1 snRNP complex

Cloning, characterization and evaluation of potent inhibitors of Shigella sonnei acetohydroxyacid synthase catalytic subunit by Won-Mook Lim; Irshad Jameel Baig; Im Joung La; Jung-Do Choi; Dong-Eun Kim; Sung-kun Kim; Jae-Wook Hyun; Giyoung Kim; Chang-Ho Kang; Young Jin Kim; Moon-Young Yoon (pp. 1825-1831).

Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate (ThDP)- and flavin adenine dinucleotide (FAD)-dependent plant and microbial enzyme that catalyzes the first common step in the biosynthesis of essential amino acids such as leucine, isoleucine and valine. To identify strong potent inhibitors against Shigella sonnei ( S. sonnei) AHAS, we cloned and characterized the catalytic subunit of S. sonnei AHAS and found two potent chemicals (KHG20612, KHG25240) that inhibit 87–93% S. sonnei AHAS activity at an inhibitor concentration of 100uM. The purified S. sonnei AHAS had a size of 65kDa on SDS-PAGE. The enzyme kinetics revealed that the enzyme has a K_m of 8.01mM and a specific activity of 0.117U/mg. The cofactor activation constant ( K_s) for ThDP and ( K_c) for Mg⁺⁺ were 0.01mM and 0.18mM, respectively. The dissociation constant ( K_d) for ThDP was found to be 0.14mM by tryptophan fluorescence quenching. The inhibition kinetics of inhibitor KHG20612 revealed an un-competitive inhibition mode with a K_ii of 2.65mM and an IC₅₀ of 9.3μM, whereas KHG25240 was a non-competitive inhibitor with a K_{ii of} 5.2mM, K_is of 1.62mM and an IC₅₀ of 12.1μM. Based on the S. sonnei AHAS homology model structure, the docking of inhibitor KHG20612 is predicted to occur through hydrogen bonding with Met 257 at a 1.7Å distance with a low negative binding energy of −9.8kcal/mol. This current study provides an impetus for the development of a novel strong antibacterial agent targeting AHAS based on these potent inhibitor scaffolds.Display Omitted► Cloned the Shigella sonnei acetohyroxyacid synthase catalytic subunit. ► Characterized the Shigella sonnei acetohyroxyacid synthase catalytic subunit. ► Identified potent inhibitors of S. sonnei AHAS; evaluated their inhibition kinetics. ► Determined the binding mode of these potent inhibitors via molecular docking.

Keywords: Abbreviations; S. sonnei; Shigella sonnei; AHAS; Acetohydroxyacid synthase; FAD; Flavin adenine dinucleotide; ThDP; Thiamine diphosphateAcetohydroxyacid synthase; Shigella sonnei; Potent chemical inhibitors; Enzyme inhibition kinetics

Diverse substrate recognition mechanism revealed by Thermotoga maritima Cel5A structures in complex with cellotetraose, cellobiose and mannotriose by Tzu-Hui Wu; Chun-Hsiang Huang; Tzu-Ping Ko; Hui-Lin Lai; Yanhe Ma; Chun-Chi Chen; Ya-Shan Cheng; Je-Ruei Liu; Rey-Ting Guo (pp. 1832-1840).

The hyperthermophilic endoglucanase Cel5A from Thermotoga maritima can find applications in lignocellulosic biofuel production, because it catalyzes the hydrolysis of glucan- and mannan-based polysaccharides. Here, we report the crystal structures in apo-form and in complex with three ligands, cellotetraose, cellobiose and mannotriose, at 1.29Å to 2.40Å resolution. The open carbohydrate-binding cavity which can accommodate oligosaccharide substrates with extensively branched chains explained the dual specificity of the enzyme. Combining our structural information and the previous kinetic data, it is suggested that this enzyme prefers β-glucosyl and β-mannosyl moieties at the reducing end and uses two conserved catalytic residues, E253 (nucleophile) and E136 (general acid/base), to hydrolyze the glycosidic bonds. Moreover, our results also suggest that the wide spectrum of Tm_Cel5A substrates might be due to the lack of steric hindrance around the C2-hydroxyl group of the glucose or mannose unit from active-site residues.Display Omitted► Cel5A can be applied to hydrolyze lignocellulose in the biofuel industry. ► The Cel5A–cellobiose and –mannotriose complex structures were determined. ► Cel5A had an open cavity for accommodating various substrates with branched chains.

Keywords: Abbreviations; BGC; β-; d; -glucose; CBI; cellobiose; CTT; cellotetraose; MAT; mannotriose; PEG; polyethyleneglycol; PCR; polymerase chain reaction; RMSD; root mean square deviation; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Tris; tris (hydroxymethyl) aminomethane; XG; xyloglucanCellulose; Cellulase; Biofuel; Crystal structure; Synchrotron radiation

Stability studies of extracellular domain two of neural-cadherin by Nagamani Vunnam; John K. McCool; Michael Williamson; Susan Pedigo (pp. 1841-1845).

Neural- (NCAD) and epithelial- (ECAD) cadherin are calcium-dependent cell-adhesive molecules, and are localized at excitatory and inhibitory synapses respectively. They play an important role in synaptogenesis, synapse maintenance and plasticity. The extracellular region plays a critical role in cadherin-mediated cell adhesion, and has five tandemly repeated ectodomains (EC1-EC5). Calcium binding is required for dimer formation between first two N-terminal domains (EC1-EC2). Despite similarity in the primary structure, the extracellular domains of NCAD and ECAD have different intrinsic stability, dimerization affinity and kinetics of disassembly. To investigate the origin of these differences, we are characterizing the modular domains individually. Here, we report studies of NCAD2, EC2 of NCAD. This domain is important for calcium binding and is the physical linkage between the dimerization interface in EC1 and the membrane proximal modular domains. Thermal-denaturation studies show that NCAD2 is less stable than ECAD2 and less influenced by the adjoining 7-residue, N- and C-terminal linker segments. In addition the NCAD2 constructs are less influenced by added salt. This difference is likely due to variation in the overall number and distribution of charges on these anionic proteins. Our studies indicate that despite their sequence similarity and apparently passive role in adhesive dimer formation, EC2 of E- and N-cadherins are distinctly different and may contribute to the differences in energetics and kinetics of dimerization.►E- and N-cadherin have different stability and calcium-dependent dimerization. ►Ectodomains were characterized individually to find the origin of these differences. ►Domain 2 for E- and N-cadherins differ in number and distribution of charges. ►Salt-dependent differences in stability correlate with differences in charge. ►Difference between E- and N-cadherin affects stability and perhaps dimerization.

Keywords: Thermal denaturation; Circular dichroism; Electrostatic repulsion

Stability studies of extracellular domain two of neural-cadherin by Nagamani Vunnam; John K. McCool; Michael Williamson; Susan Pedigo (pp. 1841-1845).

Keywords: Thermal denaturation; Circular dichroism; Electrostatic repulsion

Allosteric kinetics of the isoform 1 of human glucosamine-6-phosphate deaminase by Alvarez-Anorve Laura I. Álvarez-Añorve; Diego A. Alonzo; Rodrigo Mora-Lugo; Lara-Gonzalez Samuel Lara-González; Ismael Bustos-Jaimes; Jacqueline Plumbridge; Mario L. Calcagno (pp. 1846-1853).

The human genome contains two genes encoding for two isoforms of the enzyme glucosamine-6-phosphate deaminase (GNPDA, EC 3.5.99.6). Isoform 1 has been purified from several animal sources and the crystallographic structure of the human recombinant enzyme was solved at 1.75Å resolution (PDB ID: 1NE7). In spite of their great structural similarity, human and Escherichia coli GNPDAs show marked differences in their allosteric kinetics. The allosteric site ligand, N-acetylglucosamine 6-phosphate (GlcNAc6P), which is an activator of the K-type of E. coli GNPDA has an unusual mixed allosteric effect on hGNPDA1, behaving as a V activator and a K inhibitor ( antiergistic or crossed mixed K⁻ V⁺ effect). In the absence of GlcNAc6P, the apparent k_cat of the enzyme is so low, that GlcNAc6P behaves as an essential activator. Additionally, substrate inhibition, dependent on GlcNAc6P concentration, is observed. All these kinetic properties can be well described within the framework of the Monod allosteric model with some additional postulates. These unusual kinetic properties suggest that hGNPDA1 could be important for the maintenance of an adequate level of the pool of the UDP-GlcNAc6P, the N-acetylglucosylaminyl donor for many reactions in the cell. In this research we have also explored the possible functional significance of the C-terminal extension of hGNPDA1 enzyme, which is not present in isoform 2, by constructing and studying two mutants truncated at positions 268 and 275.►Human glucosamine-6-phosphate deaminase is an allosteric enzyme ►N-acetylglucosamine-6-phosphate produces K-inhibition and V-activation ►The net effect of this antiergistic allostery is a controlled “buffered” activation

Keywords: Abbreviations; GlcN6P; glucosamine 6-phosphate; GlcNAc6P; N-; acetylglucosamine-6-phosphate; GlcN-ol-6P; 2-amino-2-deoxy-glucitol-6-phosphate; EcGNPDA; E. coli; glucosamine 6-phosphate deaminase; hGNPDA1, hGNPDA2; human glucosamine-6-phosphate deaminase, isoforms 1 and 2,respectively; GFAT; glutamine: fructose-6-phosphate aminotransferase; SIS fold; sugar isomerase fold; MWC model; Monod–Wyman–Changeux allosteric modelHuman glucosamine-6-P deaminase; Amino sugar metabolism; Allosteric kinetics; Allosteric substrate inhibition; Antiergistic mixed; K–V; allostery

Molecular evolution and selection pressure in alpha-class carbonic anhydrase family members by Meghan E. McDevitt; Lisa A. Lambert (pp. 1854-1861).

Carbonic anhydrases (CA) are ubiquitous, and their involvement in diseases such as hypertension, diabetes, and glaucoma is well known. Most members of this family of metalloenzymes convert carbon dioxide to bicarbonate with the help of a Zn²⁺ cofactor. While the expression patterns and kinetic activities of many of these isozymes have been studied, little is known about the differences in the conservation patterns of individual residues. To better understand the molecular evolution of the CA gene family, we created multiple sequence alignments and analyzed the selection pressure (dN/dS ratios) on surface and active site residues in 248 mammalian sequences of the 14 known family members. Using the values found for amino acids of known functional importance (i.e. the three histidines that bind the zinc cofactor) as our baseline, we were able to identify other regions of possible structural and functional importance.Display Omitted► Zinc binding residues are under strong purifying selection in all CA isozymes. ► Apossible second CO₂ binding site on the surface of CA II and CA XIII is supported. ► The CA II - ICA interface likely includes His4, Phe20, Ser73, Ser166, and Gly171. ► The predicted AE1 binding site for CA II is strongly conserved. ► Potential protein interface sites for CA VI are identified.

Keywords: Carbonic anhydrase; Molecular evolution; Negative selection; Protein interaction

Molecular evolution and selection pressure in alpha-class carbonic anhydrase family members by Meghan E. McDevitt; Lisa A. Lambert (pp. 1854-1861).

Keywords: Carbonic anhydrase; Molecular evolution; Negative selection; Protein interaction

A novel approach for the characterisation of proteoglycans and biosynthetic enzymes in a snail model by Tarsis F. Gesteira; Vivien Jane Coulson-Thomas; Fernando T. Ogata; Eduardo H.C. Farias; Renan P. Cavalheiro; Marcelo A. de Lima; Gabriel L.A. Cunha; Ernesto S. Nakayasu; Igor C. Almeida; Leny Toma; Helena B. Nader (pp. 1862-1869).

Proteoglycans encompass a heterogeneous group of glycoconjugates where proteins are substituted with linear, highly negatively charged glycosaminoglycan chains. Sulphated glycosaminoglycans are ubiquitous to the animal kingdom of the Eukarya domain. Information on the distribution and characterisation of proteoglycans in invertebrate tissues is limited and restricted to a few species. By the use of multidimensional protein identification technology and immunohistochemistry, this study shows for the first time the presence and tissue localisation of different proteoglycans, such as perlecan, aggrecan, and heparan sulphate proteoglycan, amongst others, in organs of the gastropoda Achatina fulica. Through a proteomic analysis of Golgi proteins and immunohistochemistry of tissue sections, we detected the machinery involved in glycosaminoglycan biosynthesis, related to polymer formation (polymerases), as well as secondary modifications (sulphation and uronic acid epimerization). Therefore, this work not only identifies both the proteoglycan core proteins and glycosaminoglycan biosynthetic enzymes in invertebrates but also provides a novel method for the study of glycosaminoglycan and proteoglycan evolution.► Different proteoglycans in organs of the gastropoda Achatina fulica. ► Strong relationship between invertebrate and vertebrate proteoglycans. ► Novel technique of Golgi proteomics revealed conserved GAG biosynthetic machinery.

Keywords: Achatina fulica; Glycosaminoglycan; Acharan sulfate; Immunohistochemistry; Proteomic analysis

Prediction of residues involved in inhibitor specificity in the dihydrofolate reductase family by N.M. Goodey; K.G. Herbert; S.M. Hall; K.C. Bagley (pp. 1870-1879).

Dihydrofolate reductase (DHFR) is of significant recent interest as a target for drugs against parasitic and opportunistic infections. Understanding factors which influence DHFR homolog inhibitor specificity is critical for the design of compounds that selectively target DHFRs from pathogenic organisms over the human homolog. This paper presents a novel approach for predicting residues involved in ligand discrimination in a protein family using DHFR as a model system. In this approach, the relationship between inhibitor specificity and amino acid composition for sets of protein homolog pairs is examined. Similar inhibitor specificity profiles correlate with increased sequence homology at specific alignment positions. Residue positions that exhibit the strongest correlations are predicted as specificity determinants. Correlation analysis requires a quantitative measure of similarity in inhibitor specificity (S_lig) for a pair of homologs. To this end, a method of calculating S_lig values using K_I values for the two homologs against a set of inhibitors as input was developed. Correlation analysis of S_lig values to amino acid sequence similarity scores – obtained via multiple sequence alignments – was performed for individual residue alignment positions and sets of residues on 13 DHFRs. Eighteen alignment positions were identified with a strong correlation of S_lig to sequence similarity. Of these, three lie in the active site; four are located proximal to the active site, four are clustered together in the adenosine binding domain and five on the βFβG loop. The validity of the method is supported by agreement between experimental findings and current predictions involving active site residues.► Methods were developed to quantify similarity in inhibitor specificity for homologs. ► Homology-based process was developed to predict inhibitor specificity determinants. ► Eighteen DHFR positions that play a role in ligand specificity were identified. ► For these, amino acid sequence homology correlates with ligand specificity. ► Three are in active site, 5 near the active site, the rest may act allosterically.

Keywords: Abbreviations; DHFR; Dihydrofolate reductase; Hs; H. sapiens; Ec; E. coli; Mt; M. tuberculosis; Tb; T. brucei; Tg; T. gondii; Lm; L. major; Tc; T. cruzi; Pf; P. falciparum; Pv; P. vivax; Rn; R. norvegicus; Pc; P. carnii; Lc; L. casei; Ma; M. avium; MTX; Methotrexate; TMP; Trimethoprim; ZD1694; Raltitrexed; PTX; Piritrexim; LY231514; Pemetrexed; PYR; Pyrimethamine; MBP; Methylbenzoprim; CYC; Cycloguanil; TMQ; Trimetrexate; EPM; Epiroprim; CB3717; PDDFDrug-target interaction; Target prediction; Dihydrofolate reductase; Allosteric interaction; Inhibitor specificity; Specificity determinant

Prediction of residues involved in inhibitor specificity in the dihydrofolate reductase family by N.M. Goodey; K.G. Herbert; S.M. Hall; K.C. Bagley (pp. 1870-1879).

Extraordinary μs–ms backbone dynamics in Arabidopsis thaliana peroxiredoxin Q by Aden Jörgen Ådén; Marcus Wallgren; Patrik Storm; Christoph F. Weise; Alexander Christiansen; Schroder Wolfgang P. Schröder; Christiane Funk; Magnus Wolf-Watz (pp. 1880-1890).

Peroxiredoxin Q (PrxQ) isolated from Arabidopsis thaliana belongs to a family of redox enzymes called peroxiredoxins, which are thioredoxin- or glutaredoxin-dependent peroxidases acting to reduce peroxides and in particular hydrogen peroxide. PrxQ cycles between an active reduced state and an inactive oxidized state during its catalytic cycle. The catalytic mechanism involves a nucleophilic attack of the catalytic cysteine on hydrogen peroxide to generate a sulfonic acid intermediate with a concerted release of a water molecule. This intermediate is subsequently relaxed by the reaction of a second cysteine, denoted the resolving cysteine, generating an intramolecular disulfide bond and release of a second water molecule. PrxQ is recycled to the active state by a thioredoxin-dependent reduction. Previous structural studies of PrxQ homologues have provided the structural basis for the switch between reduced and oxidized conformations. Here, we have performed a detailed study of the activity, structure and dynamics of PrxQ in both the oxidized and reduced states. Reliable and experimentally validated structural models of PrxQ in both oxidation states were generated using homology based modeling. Analysis of NMR spin relaxation rates shows that PrxQ is monomeric in both oxidized and reduced states. As evident from R₂ relaxation rates the reduced form of PrxQ undergoes unprecedented dynamics on the slow μs–ms timescale. The ground state of this conformational dynamics is likely the stably folded reduced state as implied by circular dichroism spectroscopy. We speculate that the extensive dynamics is intimately related to the catalytic function of PrxQ.► The activity, structure and dynamics of the A. thaliana enzyme PrxQ were quantified. ► Validated structural models of PrxQ in both oxidation states were generated. ► PrxQ undergoes extreme µs-ms conformational dynamics in both oxidation states. ► We suggest that the observed dynamics in PrxQ is closely related to its function.

Keywords: Abbreviations; CD; circular dichroism; CPMG; Carr–Purcell–Meiboom–Gill; DTT; dithiothreitol; HSQC; heteronuclear single-quantum coherence; NMR; nuclear magnetic resonance; ROS; reactive oxygen species; TCEP; tris(2-carboxyethyl)phosphineNMR; Enzyme; Dynamics; Peroxiredoxin; Arabidopsis thaliana

Caudal-related homeodomain proteins CDX1/2 bind to DNA replication-related element binding factor by So Young Park; Mi Suk Jeong; Mi-Ae Yoo; Se Bok Jang (pp. 1891-1899).

In the intestinal epithelium, the CDX1 and CDX2 homeodomain genes play proliferative and tumor suppressor roles, respectively. The transcription factor DNA replication-related element binding factor (DREF), is an 80kDa polypeptide homodimer that plays an important role in regulating cell proliferation-related genes. Homeodomain genes encode DNA-binding proteins that play crucial roles during development by defining the body plan and determining cell fate. However, until now, the regulation of DREF function by caudal-related homeodomain proteins is poorly understood. In this study, recombinant CDX1/2 homeodomains (CDX1, amino acids [aa] 152–216 and CDX2, aa 184–248) and the DNA-binding domain of Drosophila DREF (dDREF; aa 1–125) were isolated in order to investigate the regulatory mechanism of their interaction. The expression and purification of the truncated CDX1/2 and DREF proteins were successfully performed in Escherichia coli. Models of the CDX1/2 homeodomain and dDREF were constructed using SWISS-MODEL software, a program for relative protein structure modeling. The binding of CDX1/2 and DREF proteins was detected by fluorescence measurement, size-exclusion column (SEC) chromatography, His-tagged pull-down assay, and surface plasmon resonance spectroscopy (BIAcore). In addition, we identified that four different mutants of CDX1 (S185A, N190A, T194A, and V212A) were bound to dDREF with different degrees of interaction. Our results indicate that CDX1/2 homeodomains interact with the DNA-binding domain of dDREF, thereby regulating its transcription activity.► CDX1/2 homeodomains and the DNA binding domain of dDREF were isolated. ► The binding of CDX1/2 and DREF proteins was detected. ► Structural models of the CDX1/2 HD and DREF were predicted. ► Four mutants of CDX1 were bound to DREF with different degrees of interaction.

Keywords: Homeodomain; CDX1/2; dDREF; Interaction; Mutation

Caudal-related homeodomain proteins CDX1/2 bind to DNA replication-related element binding factor by So Young Park; Mi Suk Jeong; Mi-Ae Yoo; Se Bok Jang (pp. 1891-1899).

Keywords: Homeodomain; CDX1/2; dDREF; Interaction; Mutation

Characterization of monomeric dihydrodipicolinate synthase variant reveals the importance of substrate binding in optimizing oligomerization by F. Grant Pearce; Renwick C.J. Dobson; Geoffrey B. Jameson; Matthew A. Perugini; Juliet A. Gerrard (pp. 1900-1909).

To gain insights into the role of quaternary structure in the TIM-barrel family of enzymes, we introduced mutations to the DHDPS enzyme of Thermotoga maritima, which we have previously shown to be a stable tetramer in solution. These mutations were aimed at reducing the number of salt bridges at one of the two tetramerization interface of the enzyme, which contains many more interactions than the well characterized equivalent interface of the mesophilic Escherichia coli DHDPS enzyme. The resulting variants had altered quaternary structure, as shown by analytical ultracentrifugation, gel filtration liquid chromatography, and small angle X-ray scattering, and X-ray crystallographic studies confirmed that one variant existed as an independent monomer, but with few changes to the secondary and tertiary structure. Reduction of higher order assembly resulted in a loss of thermal stability, as measured by a variety of methods, and impaired catalytic function. Binding of pyruvate increased the oligomeric status of the variants, with a concomitant increase in thermal stability, suggesting a role for substrate binding in optimizing stable, higher order structures. The results of this work show that the salt bridges located at the tetramerization interface of DHDPS play a significant role in maintaining higher order structures, and demonstrate the importance of quaternary structure in determining protein stability and in the optimization of enzyme catalysis.► Examined the role of electrostatic charges at protein interfaces. ► Removal of charged residues destabilized quaternary structure. ► Removal of charged residues reduced heat stability. ► Binding of substrate impacts quaternary structure and stability.

Keywords: Abbreviations; (; S; )-ASA; (; S; )-aspartate-semialdehyde; DHDPR; dihydrodipicolinate reductase; DHDPS; dihydrodipicolinate synthase; HTPA; (4; S; )-4-hydroxy-2,3,4,5-tetrahydro-(2; S; )-dipicolinic acid; NAL; neuraminidate lyase; rmsd; root-mean-square deviationDihydrodipicolinate synthase; (S)-lysine biosynthesis; Thermal stability; Cooperative assembly; Quaternary structure; Thermotoga maritima

Enterococcus faecalis SufU scaffold protein enhances SufS desulfurase activity by acquiring sulfur from its cysteine-153 by Gustavo P. Riboldi; Jaim S. de Oliveira; Jeverson Frazzon (pp. 1910-1918).

Iron–sulfur [Fe–S] clusters are inorganic prosthetic groups that play essential roles in all living organisms. In vivo [Fe–S] cluster biogenesis requires enzymes involved in iron and sulfur mobilization, assembly of clusters, and delivery to their final acceptor. In these systems, a cysteine desulfurase is responsible for the release of sulfide ions, which are incorporated into a scaffold protein for subsequent [Fe–S] cluster assembly. Although three machineries have been shown to be present in Proteobacteria for [Fe–S] cluster biogenesis (NIF, ISC, and SUF), only the SUF machinery has been found in Firmicutes. We have recently described the structural similarities and differences between Enterococcus faecalis and Escherichia coli SufU proteins, which prompted the proposal that SufU is the scaffold protein of the E. faecalis sufCDSUB system. The present work aims at elucidating the biological roles of E. faecalis SufS and SufU proteins in [Fe–S] cluster assembly. We show that SufS has cysteine desulfurase activity and cysteine-365 plays an essential role in catalysis. SufS requires SufU as activator to [4Fe–4S] cluster assembly, as its ortholog, IscU, in which the conserved cysteine-153 acts as a proximal sulfur acceptor for transpersulfurization reaction.► Iron-sulfur cluster biogenesis in gram-positive bacteria. ► SufU is the E. faecalis scaffold protein and reconstitutes [4Fe–4S] cluster. ► SufS is the cysteine desulfurase enzyme enclosing a catalytically cys-365. ► SufU activates E. faecalis SufS desulfurase activity. ► Transpersulfurization reaction involves cys-365 of SufS and cys-153 of SufU.

Keywords: [Fe–S] cluster biogenesis; Firmicutes; Enterococcus faecalis; suf; operon; SufU; SufS

Structural characterization of recombinant human myoglobin isoforms by¹H and¹²⁹Xe NMR and molecular dynamics simulations by Maristella Gussoni; Mariano Andrea Scorciapino; Alessandra Vezzoli; Roberto Anedda; Fulvia Greco; Matteo Ceccarelli; Mariano Casu (pp. 1919-1929).

Myoglobin (Mb), the main cytosolic oxygen storage/deliver protein, is also known to interact with different small ligands exerting other fundamental physiological roles. In Humans up to five different Mb isoforms are present. The two most expressed ones (>90%) differ only at the 54th position, K54 (Mb-I) and E54 (Mb-II) respectively. High-altitude populations are characterized by a higher Mb concentration in skeletal muscle, totally attributable to Mb-II, as well as a higher efficiency of locomotion, leading to the hypothesis of a cause–effect relationship with the evolutionary response to the high-altitude hypoxic environment. In this work, a first structural characterization of the two more expressed human Mb isoforms has been carried out. In particular, a detailed¹H and¹²⁹Xe NMR study was aimed to characterize the structure of the hydrophobic cavities around the heme group. Experimental results have been compared to those from MD simulations, i.e. volume fluctuations and occurrence. Electronic structure of the heme ring ground state resulted to be comparable for the two investigated isoforms, despite the single point mutation at position 54. However, the use of¹²⁹Xe as a probe revealed small but significant modifications in the structure of internal cavities. MD simulations supported NMR results indicating interesting structural/dynamical differences in the average volume and occurrence of the main cavities lining Mb prosthetic group.► We begin a structural characterization of the two more expressed human Mb isoforms. ►¹H and¹²⁹Xe NMR study allows structural characterization of the main binding sites. ► A general similarity for the ground state electronic structure of the heme is shown. ► NMR and MD support subtle differences in cavities around the heme region. ► Different structural/dynamical behavior for these two proteins can be suggested.

Keywords: Abbreviations; NMR; Nuclear Magnetic Resonance; MD; Molecular Dynamics; Mb; myoglobin; deoxy-Mb; deoxy myoglobin; CN-Mb; cyano myoglobin; met-Mb; met myoglobin; DSS; 2,2-dimethyl-2-silapentane-5-sulfonate; PBS; potassium phosphate buffered solution¹; H-NMR; ¹²⁹; Xe-NMR; Myoglobin; Xenon; Xenon cavity; Molecular dynamics

Molecular mechanisms of the anomalous thermal aggregation of green fluorescent protein by Bogan S. Melnik; Nikolay V. Molochkov; Dmitry A. Prokhorov; Vladimir N. Uversky; Viktor P. Kutyshenko (pp. 1930-1939).

The peculiarities of thermal denaturation and interaction with water of the cycle-3 mutant of green fluorescent protein (GFP) were analyzed by NMR techniques and compared with those of bovine carbonic anhydrase II (BCA-II). Irreversible thermal denaturation was accompanied by massive GFP aggregation with no detectable accumulation of soluble denatured protein. Analysis of the spin diffusion data suggested that the internal part of the GFP β-can is involved in intensive interactions with water molecules. As a result, at high temperatures, the GFP structure does not unfold but rather breaks, consequently leading to enhanced protein aggregation. This is very different from typical BCA-II behavior.► Thermal denaturation behavior of GFP is remarkably different from that of BCA-II. ► The inside of the GFP β-can is involved in intensive interactions with water molecules. ► At high temperatures, the GFP structure does not unfold but breaks. ► Irreversible thermal denaturation GFP is accompanied by massive aggregation. ► No accumulation of soluble denatured protein is detected.

Keywords: Abbreviations; SD; spin diffusion; SSD; spectrum of spin diffusion; 1M-spectrum; reference; ¹; H NMR spectrum; APS; associate of protein and solvent molecules; offRI; off-resonance irradiation; NMR; nuclear magnetic resonance; BCA-II; bovine carbonic anhydrase II; GFP; green fluorescent protein; EBFP; enhanced blue fluorescent protein; ECFP; enhanced cyan fluorescent protein; EGFP; enhanced green fluorescent protein; EYFP; enhanced yellow fluorescent protein; DsRed; red fluorescent protein; FRET; fluorescence resonance energy transfer; GdmCl; guanidinium chlorideGreen fluorescent protein; Carbonic anhydrase II; NMR; Spin diffusion; Protein association; Protein–solvent interaction

Thermodynamic analysis of ionizable groups involved in the catalytic mechanism of human matrix metalloproteinase 7 (MMP-7) by Hitoshi Takeharu; Kiyoshi Yasukawa; Kuniyo Inouye (pp. 1940-1946).

Human matrix metalloproteinase 7 (MMP-7) exhibits a broad bell-shaped pH-dependence with the acidic and alkaline p K_e (p K_e1 and p K_e2) values of about 4 and 10. In this study, we estimated the ionizable groups involved in its catalytic mechanism by thermodynamic analysis. p K_a of side chains ofL-Asp,L-Glu, L-His,L-Cys,L-Tyr,L-Lys, andL-Arg at 25–45°C were determined by the pH titration of amino-acid solutions, from which their enthalpy changes, ∆H°, of deprotonation were calculated. p K_e1 and p K_e2 of MMP-7 at 15–45°C were determined in the hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[ N³-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH₂, from which ∆H^o for p K_e1 and p K_e2 was calculated. The ∆H^o for p K_e1 (−20.6±6.1kJmol−¹) was similar to that forL-Glu (−23.6±5.8kJmol−¹), and the ∆H^o for p K_e2 (89.9±4.0kJmol−¹) was similar to those forL-Arg (87.6±5.5kJmol−¹) andL-Lys (70.4±4.4kJmol−¹). The mutation of the active-site residue Glu198 into Ala completely abolished the activity, suggesting that Glu198 is the ionizable group for p K_e1. On the other hand, no arginine or lysine residues are found in the active site of MMP-7. We proposed a possibility that a protein-bound water is the ionizable group for p K_e2.► MMP-7 activity exhibits a broad bell-shaped pH-dependence with the p K_e1 and p K_e2 values of 4 and 10. ► The p K_e2 value suggests Tyr or Lys, but no Lys is in the active site, and thus Tyr219 is the candidate. ► However, Tyr219 was declined to be the p K_e2 residue by site-directed mutagenesis analysis of MMP-7. ► Thermodynamic analysis suggested that Glu198 is responsible for p K_e1. ► It also suggested that a protein-bound water molecule is responsible p K_e2.

Keywords: Abbreviations; AMPSO; 3-[(1,1-dimethyl-2-hydroxy-ethyl)amino]-2-hydroxypropane sulfonic acid; DMSO; dimethyl sulfoxide; HEPES; 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid; K; _e; proton dissociation constant; MES; 2-(; N; -morpholino)ethanesulfonic acid; MMP; matrix metalloproteinase; MOCAc-PLG; (7-methoxycoumarin-4-yl)acetyl-; L; -Pro-; L; -Leu-Gly; MOCAc- PLGL(Dpa)AR; (7-methoxycoumarin-4-yl)acetyl-; L; -Pro-; L; -Leu-Gly-; L; -Leu-[; N; ³; -(2,4-dinitrophenyl)-; L; -2,3-diaminopropionyl]-; L; -Ala-; L; -Arg-NH; ₂Ionizable group; Matrix metalloproteinase; MMP-7; Proton dissociation constant; Thermodynamic analysis

Thermodynamic analysis of ionizable groups involved in the catalytic mechanism of human matrix metalloproteinase 7 (MMP-7) by Hitoshi Takeharu; Kiyoshi Yasukawa; Kuniyo Inouye (pp. 1940-1946).

Purification and kinetic characterization of human indoleamine 2,3-dioxygenases 1 and 2 (IDO1 and IDO2) and discovery of selective IDO1 inhibitors by David Meininger; Leeanne Zalameda; Yichin Liu; Lara P. Stepan; Luis Borges; John D. McCarter; Claire L. Sutherland (pp. 1947-1954).

Indoleamine 2,3-dioxygenase (IDO1) catalyzes the first step in tryptophan breakdown along the kynurenine pathway. Therapeutic inhibition of IDO1 is receiving much attention due to its proposed role in the pathogenesis of several diseases including cancer, hypotension and neurodegenerative disorders. A related enzyme, IDO2 has recently been described. We report the first purification and kinetic characterization of human IDO2 using a facilel-tryptophan consumption assay amenable to high throughput screening. We found that the K_m of human IDO2 forl-tryptophan is much higher than that of IDO1. We also describe the identification and characterization of a new IDO1 inhibitor compound, Amg-1, by high throughput screening, and compare the inhibition profiles of IDO1 and IDO2 with Amg-1 and previously described compounds. Our data indicate that human IDO1 and IDO2 have different kinetic parameters and different inhibition profiles. Docking of Amg-1 and related analogs to the known structure of IDO1 and to homology-modeled IDO2 suggests possible rationales for the different inhibition profiles of IDO1 and IDO2.► IDO is an enzyme involved in cancer, hypotension and neurodegenerative disease. ► We report the first purification and kinetic characterization of human IDO2. ► Human IDO1 and IDO2 have different kinetic parameters and inhibition profiles. ► We describe the discovery of a new IDO1-selective inhibitor compound. ► IDO1 and IDO2 may have different functions and can be selectively targeted.

Keywords: Abbreviations; IDO; indoleamine 2,3-dioxygenase; Trp; l; -tryptophan; TDO; tryptophan 2,3-dioxygenase; 1MT; 1-methyl; l; -tryptophan; MTH; methyl thiohydantoin; PTH; phenylthiohydantoinHuman; IDO1; IDO2; Kinetics; Inhibition

Design of Factor XIII V34X activation peptides to control ability to interact with thrombin mutants by Madhavi A. Jadhav; R. Cory Lucas; Whitney N. Goldsberry; Muriel C. Maurer (pp. 1955-1963).

Thrombin helps to activate Factor XIII (FXIII) by hydrolyzing the R37-G38 peptide bond. The resultant transglutaminase introduces cross-links into the fibrin clot. With the development of therapeutic coagulation factors, there is a need to better understand interactions involving FXIII. Such knowledge will help predict ability to activate FXIII and thus ability to promote/hinder the generation of transglutaminase activity. Kinetic parameters have been determined for a series of thrombin species hydrolyzing the FXIII (28–41) V34X activation peptides (V34, V34L, V34F, and V34P). The V34P substitution introduces PAR4 character into the FXIII, and the V34F exhibits important similarities to the cardioprotective V34L. FXIII activation peptides containing V34, V34L, or V34P could each be accommodated by alanine mutants of thrombin lacking either the W60d or Y60a residue in the 60-insertion loop. By contrast, FXIII V34F AP could be cleaved by thrombin W60dA but not by Y60aA. FXIII V34P is highly reliant on the thrombin W215 platform for its strong substrate properties whereas FXIII V34F AP becomes the first segment that can maintain its K_m upon loss of the critical thrombin W215 residue. Interestingly, FXIII V34F AP could also be readily accommodated by thrombin L99A and E217A. Hydrolysis of FXIII V34F AP by thrombin W217A/E217A (WE) was similar to that of FXIII V34L AP whereas WE could not effectively cleave FXIII V34P AP. FXIII V34F and V34P AP show promise for designing FXIII activation systems that are either tolerant of or greatly hindered by the presence of anticoagulant thrombins.► FXIII V34F and V34P AP show promise for controlling thrombin activation of FXIII. ► FXIII V34F AP is highly reliant on thrombin Y60a and FXIII V34P on thrombin W215. ► FXIII V34P AP with its PAR4 character promotes best binding to wild-type thrombin. ► FXIII V34F AP is first activation peptide that accommodates loss of thrombin W215. ► FXIII V34F and cardioprotective V34L AP exhibit similar kinetics with thrombin WE.

Keywords: Abbreviations; FXIII (28–41) AP; blood clotting Factor XIII Activation Peptide; FXIII V34 AP; wild type Factor XIII Activation Peptide; FXIII V34L AP; Val to Leu polymorphism of Factor XIII Activation Peptide; FXIII V34F AP; Val to Phe substitution; FXIII V34P AP; Val to Pro substitution; IIa; thrombin; ABE-I; anion-binding exosite-I; ABE-II; anion-binding exosite-II; Fbg; fibrinogen; PC; protein C; PAR1; protease activated receptor 1; PAR4; protease activated receptor 4Factor XIII; Transglutaminase; Thrombin; Fibrinogen; Kinetic; Coagulation

Design of Factor XIII V34X activation peptides to control ability to interact with thrombin mutants by Madhavi A. Jadhav; R. Cory Lucas; Whitney N. Goldsberry; Muriel C. Maurer (pp. 1955-1963).

Proteomic analyses of apoplastic proteins from germinating Arabidopsis thaliana pollen by Weina Ge; Yun Song; Cuijun Zhang; Yafang Zhang; Alma L. Burlingame; Yi Guo (pp. 1964-1973).

Pollen grains play important roles in the reproductive processes of flowering plants. The roles of apoplastic proteins in pollen germination and in pollen tube growth are comparatively less well understood. To investigate the functions of apoplastic proteins in pollen germination, the global apoplastic proteins of mature and germinated Arabidopsis thaliana pollen grains were prepared for differential analyses by using 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE) saturation labeling techniques. One hundred and three proteins differentially expressed ( p value≤0.01) in pollen germinated for 6h compared with un-germination mature pollen, and 98 spots, which represented 71 proteins, were identified by LC–MS/MS. By bioinformatics analysis, 50 proteins were identified as secreted proteins. These proteins were mainly involved in cell wall modification and remodeling, protein metabolism and signal transduction. Three of the differentially expressed proteins were randomly selected to determine their subcellular localizations by transiently expressing YFP fusion proteins. The results of subcellular localization were identical with the bioinformatics prediction. Based on these data, we proposed a model for apoplastic proteins functioning in pollen germination and pollen tube growth. These results will lead to a better understanding of the mechanisms of pollen germination and pollen tube growth.Display Omitted► Differential proteomics analysis of germinating pollen of Arabidopsis by2D DIGE. ► 103 spots differentially expressed ( p value≤0.01) were identified by LC–MS/MS. ► 98 spots were represented and 71 proteins were identified. ► Subcellular localization of three proteins has been confirmed by YFP fusion proteins. ► A model of apoplastic proteins function in pollen germination was proposed.

Keywords: Arabidopsis thaliana; Pollen germination; Apoplast; 2-D DIGE; Proteomic

Proteomic analyses of apoplastic proteins from germinating Arabidopsis thaliana pollen by Weina Ge; Yun Song; Cuijun Zhang; Yafang Zhang; Alma L. Burlingame; Yi Guo (pp. 1964-1973).

Keywords: Arabidopsis thaliana; Pollen germination; Apoplast; 2-D DIGE; Proteomic

Catalytic oxidation of o-aminophenols and aromatic amines by mushroom tyrosinase by Munoz-Munoz Jose Luis Muñoz-Muñoz; Francisco Garcia-Molina; Pedro Antonio Garcia-Ruiz; Ramon Varon; Jose Tudela; Jose N. Rodriguez-Lopez; Francisco Garcia-Canovas (pp. 1974-1983).

The kinetics of tyrosinase acting on o-aminophenols and aromatic amines as substrates was studied. The catalytic constants of aromatic monoamines and o-diamines were both low, these results are consistent with our previous mechanism in which the slow step is the transfer of a proton by a hydroxyl to the peroxide in oxy-tyrosinase (Fenoll et al., Biochem. J. 380 (2004) 643–650). In the case of o-aminophenols, the hydroxyl group indirectly cooperates in the transfer of the proton and consequently the catalytic constants in the action of tyrosinase on these compounds are higher. In the case of aromatic monoamines, the Michaelis constants are of the same order of magnitude than for monophenols, which suggests that the monophenols bind better (higher binding constant) to the enzyme to facilitate the π–π interactions between the aromatic ring and a possible histidine of the active site. In the case of aromatic o-diamines, both the catalytic and Michaelis constants are low, the values of the catalytic constants being lower than those of the corresponding o-diphenols. The values of the Michaelis constants of the aromatic o-diamines are slightly lower than those of their corresponding o-diphenols, confirming that the aromatic o-diamines bind less well (lower binding constant) to the enzyme.► We have demonstrated that mushroom TYR oxidises aromatic monoamines and diamines and aminophenols. ► We propose structural and kinetic mechanisms that agree with the experimental results obtained. ► We have kinetically characterised a wide number of substrates.

Keywords: Tyrosinase; Aromatic amine; Aromatic; o; -diamines; o; -aminophenols; Catalytic constant; Michaelis constant

Glass transition and dynamics in BSA–water mixtures over wide ranges of composition studied by thermal and dielectric techniques by A. Panagopoulou; A. Kyritsis; R. Sabater i Serra; Gomez Ribelles J.L. Gómez Ribelles; N. Shinyashiki; P. Pissis (pp. 1984-1996).

Protein–water dynamics in mixtures of water and a globular protein, bovine serum albumin (BSA), was studied over wide ranges of composition, in the form of solutions or hydrated solid pellets, by differential scanning calorimetry (DSC), thermally stimulated depolarization current technique (TSDC) and dielectric relaxation spectroscopy (DRS). Additionally, water equilibrium sorption isotherm (ESI) measurements were performed at room temperature. The crystallization and melting events were studied by DSC and the amount of uncrystallized water was calculated by the enthalpy of melting during heating. The glass transition of the system was detected by DSC for water contents higher than the critical water content corresponding to the formation of the first sorption layer of water molecules directly bound to primary hydration sites, namely 0.073 (grams of water per grams of dry protein), estimated by ESI. A strong plasticization of the T_g was observed by DSC for hydration levels lower than those necessary for crystallization of water during cooling, i.e. lower than about 0.3 (grams of water per grams of hydrated protein) followed by a stabilization of T_g at about −80°C for higher water contents. The α relaxation associated with the glass transition was also observed in dielectric measurements. In TSDC a microphase separation could be detected resulting in double T_g for some hydration levels. A dielectric relaxation of small polar groups of the protein plasticized by water, overlapped by relaxations of uncrystallized water molecules, and a separate relaxation of water in the crystallized water phase (bulk ice crystals) were also recorded.Display Omitted► Thermal and dielectric studies of BSA–water mixtures. ► Studies of water and protein dynamics over extremely wide ranges of water content. ► Evolution of dynamics with hydration level. ► Determination of critical water contents. ► Assignment of relaxations to particular origins.

Keywords: Molecular mobility; Hydrated protein; Glass transition; Uncrystallized water; Dielectric relaxation; Plasticization

Intraprotein electron transfer between the FMN and heme domains in endothelial nitric oxide synthase holoenzyme by Changjian Feng; Valentina Taiakina; Dipak K. Ghosh; J. Guy Guillemette; Gordon Tollin (pp. 1997-2002).

Intraprotein electron transfer (IET) from flavin mononucleotide (FMN) to heme is an essential step in nitric oxide (NO) synthesis by NO synthase (NOS). The IET kinetics in neuronal and inducible NOS (nNOS and iNOS) holoenzymes have been previously determined in our laboratories by laser flash photolysis [reviewed in: C.J. Feng, G. Tollin, Dalton Trans., (2009) 6692–6700]. Here we report the kinetics of the IET in a bovine endothelial NOS (eNOS) holoenzyme in the presence and absence of added calmodulin (CaM). The IET rate constant in the presence of CaM is estimated to be ~4.3s⁻¹. No IET was observed in the absence of CaM, indicating that CaM is the primary factor in controlling the FMN−heme IET in the eNOS enzyme. The IET rate constant value for the eNOS holoenzyme is approximately 10 times smaller than those obtained for the iNOS and CaM-bound nNOS holoenzymes. Possible mechanisms underlying the difference in IET kinetics among the NOS isoforms are discussed. Because the rate-limiting step in the IET process in these enzymes is the conformational change from input state to output state, a slower conformational change (than in the other isoforms) is most likely to cause the slower IET in eNOS.► The FMN-heme IET (interdomain electron transfer) is essential for NOS function. ► The IET kinetics in an eNOS holoenzyme was directly measured by CO photolysis. ► The IET was only observed with added calmodulin. ► The eNOS IET rate value is ~10 times smaller than other NOS isoforms.

Keywords: Abbreviations; NO; nitric oxide; NOS; nitric oxide synthase; eNOS; endothelial NOS; iNOS; inducible NOS; nNOS; neuronal NOS; CaM; calmodulin; IET; intraprotein interdomain electron transfer; FMN; flavin mononucleotide; FMNH; ^•; FMN semiquinone; FMN; _hq; FMN hydroquinone; FAD; flavin adenine dinucleotide; FADH; ^•; FAD semiquinone; FAD; _hq; FAD hydroquinone; dRF; 5-deazariboflavin; dRFH; ^•; 5-deazariboflavin semiquinone; H; ₄; B; (6R)-5,6,7,8-tetrahydrobiopterin; l; -Arg; l; -arginineNitric oxide synthase; Kinetics; Electron transfer; Mechanism

Correlating crosslink formation with enzymatic activity in cysteine dioxygenase by Eleni Siakkou; Malcolm T. Rutledge; Sigurd M. Wilbanks; Guy N.L. Jameson (pp. 2003-2009).

Cysteine dioxygenase (CDO) from rat and other mammals exhibits a covalent post-translational modification between the residues C93 and Y157 that is in close proximity to the active site, and whose presence enhances the enzyme's activity. Protein with and without C93-Y157 crosslink migrates as distinct bands in SDS-PAGE, allowing quantification of the relative ratios between the two forms by densitometry of the respective bands. Expression of recombinant rat wild type CDO in Escherichia coli typically produces 40–50% with the C93-Y157 crosslink. A strategy was developed to increase the ratio of the non-crosslinked form in an enzyme preparation of reasonable quantity and purity, allowing direct assessment of the activity of non-crosslinked CDO and mechanism of formation of the crosslink. The presence of ferrous iron and oxygen is a prerequisite for C93-Y157 crosslink formation. Absence of oxygen during protein expression increased the fraction of non-crosslinked CDO, while presence of the metal chelator EDTA had little effect. Metal affinity chromatography was used to enrich non-crosslinked content. Both the enzymatic rate of cysteine oxidation and the amount of cross-linking between C93 and Y157 increased significantly upon exposure of CDO to air/oxygen and substrate cysteine in the presence of iron in a hitherto unreported two-phase process. The instantaneous activity was proportional to the amount of crosslinked enzyme present, demonstrating that the non-crosslinked form has negligible enzymatic activity. The biphasic kinetics suggest the existence of an as yet uncharacterised intermediate in crosslink formation and enzyme activation.► Anaerobic expression of mainly non-crosslinked cysteine dioxygenase. ► Separation of different ratios crosslinked/non-crosslinked enzyme using IMAC. ► Mainly non-crosslinked enzyme shows low initial activity. ► Enzyme activity increases with crosslink formation.

Keywords: Cysteine dioxygenase; Post-translational modification; Enzyme activity; Anaerobic expression

Correlating crosslink formation with enzymatic activity in cysteine dioxygenase by Eleni Siakkou; Malcolm T. Rutledge; Sigurd M. Wilbanks; Guy N.L. Jameson (pp. 2003-2009).

Keywords: Cysteine dioxygenase; Post-translational modification; Enzyme activity; Anaerobic expression

Corrigendum to “Functional proteomics reveal the effect of Salvia miltiorrhiza aqueous extract against vascular atherosclerotic lesions” [Biochim. Biophys. Acta 1804 (2010) 1310–1321] by Yu-Chiang Hung; Pei-Wen Wang; Tai-Long Pan (pp. 2010-2010).

Corrigendum to “Surface localized and extracellular glyceraldehyde-3-phosphate dehydrogenase of Bacillus anthracis is a plasminogen binding protein” [Biochem. Biophys. Acta 1804 (2010) 2111–2120] by Sumit Kumar Matta; Shivangi Agarwal; Rakesh Bhatnagar (pp. 2011-2011).

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BBA - Proteins and Proteomics (v.1814, #12)