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BBA - Proteins and Proteomics (v.1764, #4)
Efficient solubilization buffers for two-dimensional gel electrophoresis of acidic and basic proteins extracted from wheat seeds by Gurusamy Chinnasamy; Christof Rampitsch (pp. 641-644).
Plant tissues are made up of a broad range of proteins with a variety of properties. After extraction, solubilization of a diverse range of plant proteins for efficient proteomic analysis using two-dimensional electrophoresis is a challenging process. We tested the efficiency of 12 solubilization buffers in dissolving acidic and basic proteins extracted from mature seeds of wheat. The buffer containing two chaotropes (urea and thiourea), two detergents (3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate and N-decyl- N, N-dimethyl-3-ammonio-1-propane-sulfonate), two reducing agents (dithiothreitol and tris (2-carboxyethyl) phosphine hydrochloride) and two types of carrier ampholytes (BioLyte pH 4–6 and pH 3–10) solubilized the most acidic proteins in the pH range between 4 and 7. The buffer made up of urea, thiourea, 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate, DeStreak reagent (Amersham Biosciences, Uppsala, Sweden) and immobilized pH gradient buffer, pH 6–11 (Amersham Biosciences) solubilized the most basic proteins in the pH range between 6 and 11. These two buffers produced two-dimensional gels with high resolution, superior quality and maximum number of detectable protein (1425 acidic protein and 897 basic protein) spots.
Keywords: Abbreviations; SB3-10; N; -decyl-; N; ,; N; -dimethyl-3-ammonio-1-propane-sulfonate; TCEP-HCl; Tris (2-carboxyethyl) phosphine hydrochlorideAcidic and basic protein; Isoelectric focusing; Protein solubilization; Two-dimensional electrophoresis; Wheat seed
Functional interaction of nitrogenous organic bases with cytochrome P450: A critical assessment and update of substrate features and predicted key active-site elements steering the access, binding, and orientation of amines by Peter Hlavica (pp. 645-670).
The widespread use of nitrogenous organic bases as environmental chemicals, food additives, and clinically important drugs necessitates precise knowledge about the molecular principles governing biotransformation of this category of substrates. In this regard, analysis of the topological background of complex formation between amines and P450s, acting as major catalysts in C- and N-oxidative attack, is of paramount importance. Thus, progress in collaborative investigations, combining physico-chemical techniques with chemical-modification as well as genetic engineering experiments, enables substantiation of hypothetical work resulting from the design of pharmacophores or homology modelling of P450s. Based on a general, CYP2D6-related construct, the majority of prospective amine-docking residues was found to cluster near the distal heme face in the six known SRSs, made up by the highly variant helices B′, F and G as well as the N-terminal portion of helix C and certain β-structures. Most of the contact sites examined show a frequency of conservation <20%, hinting at the requirement of some degree of conformational versatility, while a limited number of amino acids exhibiting a higher level of conservation reside close to the heme core. Some key determinants may have a dual role in amine binding and/or maintenance of protein integrity. Importantly, a series of non-SRS elements are likely to be operative via long-range effects. While hydrophobic mechanisms appear to dominate orientation of the nitrogenous compounds toward the iron-oxene species, polar residues seem to foster binding events through H-bonding or salt-bridge formation. Careful uncovering of structure–function relationships in amine–enzyme association together with recently developed unsupervised machine learning approaches will be helpful in both tailoring of novel amine-type drugs and early elimination of potentially toxic or mutagenic candidates. Also, chimeragenesis might serve in the construction of more efficient P450s for activation of amine drugs and/or bioremediation.
Keywords: Abbreviations; ABT; 1-aminobenzotriazole; AMAP; N; -(; p; -azidobenzyl)-; N; -methyl-; p; -aminophenethylamine; AMPI; 3-amino-5-methoxy-2-(pyridylmethyl)indenone; BBT; N; -benzyl-1-aminobenzotriazole; BMAC; N; -butyl-7-methoxy-4-(aminomethyl)coumarin; CPI; 4-(4-chlorophenyl)imidazole; DTF; density functional theory; CEA; 1-cyclohexylethylamine; DPI; 4,5-diphenylimidazole; FSSP; fold and secondary structure in proteins; Glu-P-1; 2-amino-6-methyldipyrido[1,2-; a; : 3′,2′-; d; ]imidazole; IMI; imidazolylmethyleneindane; IQ; 2-amino-3-methylimidazo[4,5-; f; ]quinoline; MAMC; 7-methoxy-4-(aminomethyl)coumarin; MBA; N; -(α-methylbenzyl)-1-aminobenzotriazole; MD; molecular dynamics; MDMA; 3,4-methylenedioxyamphetamine; MeIQ; 2-amino3,5-dimethylimidazo[4,5-; f; ]quinoline; MPEA; 4-methoxyphenethylamine; MPTP; 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NNK; 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; P450 (CYP); cytochrome P450; PhIP; 2-amino-1-methyl-6-phenylimidazo[4,5-; b; ]pyridine; QSAR; quantitative structure–activity relationship; RMSD; root-mean-square deviation; SRS; substrate recognition site; Trp-P-1; 3-amino-1,4-dimethyl-; 5H; -pyrido[4,3-; b; ]indoleCytochrome P450; Basic amine; Docking site
Identification and characterization of glucoamylase from the fungus Thermomyces lanuginosus by Thor S. Thorsen; Anders H. Johnsen; Knud Josefsen; Bo Jensen (pp. 671-676).
The glucoamylase from the thermophilic fungus Thermomyces lanuginosus has a molecular weight of 66 kDa and was characterized with isoelectric point, pH and temperature optimum of 3.8–4.0, 5.0 and 70 °C, respectively. In addition, the activation energy is 60.4 kJ/mol, Km is 3.5 mM and kcat is 25.3 s−1. The glucoamylase was partially sequenced on the protein level, and the complete glucoamylase gene including its promoter (but excluding its terminator region) was cloned and sequenced. The glucoamylase protein comprises 617 amino acid residues and shows 60% identity with the glucoamylase from the thermophilic fungus Talaromyces emersonii. cDNA encoding Thermomyces lanuginosus glucoamylase was expression cloned into Pichia pastoris, producing approximately 7.4 U/ml. It was concluded that alternative mRNA splicing as it might occur in Aspergillus niger glucoamylase is not responsible for the occurrence of different glucoamylase isoforms in Thermomyces lanuginosus.
Keywords: Glucoamylase; Thermomyces lanuginosus; Cloning; Enzyme; Thermostability
Temperature dependence of chaperone-like activity and oligomeric state of αB-crystallin by Francesco Spinozzi; Paolo Mariani; Franco Rustichelli; Heinz Amenitsch; Federico Bennardini; Giovanni Maria Mura; Alessio Coi; Maria Luisa Ganadu (pp. 677-687).
The chaperone-like activity and the oligomeric state of αB-crystallin were studied at different temperatures and in the presence of urea and thiocyanate. The activity, assessed measuring the ability of αB-crystallin to prevent the aggregation of denatured insulin, strongly depends on temperature. While a significant activity increase was detected at 42 °C, the presence of urea and thiocyanate does not affect the protein activity in an irreversible way. In-solution SAXS measurements performed in the same experimental conditions showed that αB-crystallin forms near-spherical, hollowed, polydisperse oligomers, whose dimensions change above 42 °C. Moreover, in the presence of urea and thiocyanate, a global fit analysis confirms the high stability of αB-crystallin assemblies in relationship with their variable quaternary structure. In particular, the changes in the inner radius as well as the thickness and dispersion of the protein shell, account for the preservation of the chaperone-like activity.
Keywords: αB-crystallin; SAXS; Chaperone activity; Heat shock protein; Protein aggregation; Quaternary structure
Structural elements in dextran glucosidase responsible for high specificity to long chain substrate by Wataru Saburi; Haruhide Mori; Saori Saito; Masayuki Okuyama; Atsuo Kimura (pp. 688-698).
Dextran glucosidase from Streptococcus mutans (SMDG) and Bacillus oligo-1,6-glucosidases, members of glycoside hydrolase family 13 enzymes, have the high sequence similarity. Each of them is specific to α-1,6-glucosidic linkage at the non-reducing end of substrate to liberate glucose. The activities toward long isomaltooligosaccharides were different in both enzymes, in which SMDG and oligo-1,6-glucosidase showed high and low activities, respectively. We determined the structural elements essential for high activity toward long-chain substrate. From conformational comparison between SMDG and B. cereus oligo-1,6-glucosidase (three-dimensional structure has been solved), Trp238 and short β→α loop 4 of SMDG were considered to contribute to the high activity to long-chain substrate. W238A had similar kcat/ Km value for isomaltotriose to that for isomaltose, suggesting that the affinity of subsite +2 was decreased by Trp238 replacement. Trp238 mutants as well as the chimeric enzyme having longer β→α loop 4 of B. subtilis oligo-1,6-glucosidase showed lower preference for long-chain substrates, indicating that both Trp238 and short β→α loop 4 were important for high activity to long-chain substrates.
Keywords: Abbreviations; SMDG; Streptococcus mutans; dextran glucosidase; GH family; glycoside hydrolase family; BCOG; Bacillus cereus; oligo-1,6-glucosidase; AS; Neisseria polysaccharea; amylosucrase; CD; circular dichroism; CGTase; cyclodextrin glucanotransferase; TVAII; Thermoactinomyces vulgaris; R-47 α-amylase II; p; NPG; p; -nitrophenyl α-glucoside; p; NP; p; -nitrophenol; DP; degree of polymerization; IGn; isomaltooligosaccharides with DPnDextran glucosidase; Site-directed mutagenesis; Substrate specificity; α-amylase; α-glucosidase; Glycoside hydrolase family 13
Efficiency of exonucleolytic action of apurinic/apyrimidinic endonuclease 1 towards matched and mismatched dNMP at the 3′ terminus of different oligomeric DNA structures correlates with thermal stability of DNA duplexes by Nadezhda S. Dyrkheeva; Alexander A. Lomzov; Dmitriy V. Pyshnyi; Svetlana N. Khodyreva; Olga I. Lavrik (pp. 699-706).
Human DNA apurinic/apyrimidinic endonuclease 1 (APE1) is involved in the DNA base excision repair process. In addition to its AP (apurinic/apyrimidinic) endonucleolytic function, APE1 possesses 3′ phosphodiesterase and 3′–5′ exonuclease activities. The 3′–5′ exonuclease activity is considered important in proofreading of DNA synthesis catalyzed by DNA polymerase β. Here, we examine the removal of matched and mismatched dNMP from the 3′ terminus of the 3′-recessed and nicked DNA by the APE1 activity using two different reaction buffers. To investigate whether the ability of APE1 to excise nucleotides from the 3′ terminus depends on the thermal stability of the DNA duplex, we studied this characteristic of the DNAs that were used in the exonuclease assays in these two buffers. Our data confirm that APE1 removes mismatched nucleotides from the 3′ terminus of DNA more efficiently than matched pairs. Both the efficiency of the 3′–5′ exonuclease activity of APE1 and the thermal stability of DNA duplexes varied depending on the nature of the flanking group at the 5′ margin of the nick. The 3′–5′ exonuclease activity of APE1 shows a preference for substrates with a hydroxyl group at the 5′ margin of the nick as well as for flapped and recessed DNAs.
Keywords: 3′–5′ exonuclease activity of APE1; Base excision repair; Proofreading; Thermal stability of DNA duplex; DNA mismatch
Isolation and characterization of an apoptotic and platelet aggregation inhibitingl-amino acid oxidase from Vipera berus berus (common viper) venom by Mari Samel; Heiki Vija; Gunilla Rönnholm; Jüri Siigur; Nisse Kalkkinen; Ene Siigur (pp. 707-714).
Anl-amino acid oxidase was isolated from the venom of the common viper Vipera berus berus by a three-step procedure combining gel filtration, ion exchange and hydrophobic chromatography. The enzyme is a non-covalently bound homodimer with a monomeric molecular mass of 57.7 kDa. The N-terminal amino acid sequence and the internal peptide sequences show close structural homology with other snake venoml-amino acid oxidases. The purified protein catalyzed oxidative desamination ofl-amino acids, the most specific substrate isl-Phe. The best substrates among the studied 20 amino acids were:l-Met,l-Leu,l-Phe,l-Ile,l-Arg andl-His. Five amino acids,l-Ser,l-Pro, Gly,l-Thr andl-Cys, were not oxidized. The enzyme inhibited ADP-induced platelet aggregation dose-dependently with an IC50 of 0.07 μM. The effect was neutralized by catalase. V. berus berus LAAO induced apoptosis in cultured HeLa and K562 cells as shown by DNA fragmentation gel pattern. The induction of apoptosis was inhibited by catalase.
Keywords: Abbreviations; LAAO; l; -amino acid oxidase; FAD; flavine adenine dinucleotide; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PRP; platelet-rich plasma; MALDI-TOF MS; matrix-assisted laser desorption/ionization time-of-flight mass spectrometrySnake venom; Vipera berus berus; l; -amino acid oxidase; Apoptosis; Platelet aggregation; Partial sequencing
Molecular and catalytic properties of a novel cytochrome c nitrite reductase from nitrate-reducing haloalkaliphilic sulfur-oxidizing bacterium Thioalkalivibrio nitratireducens by Tamara V. Tikhonova; Alvira Slutsky; Alexey N. Antipov; Konstantin M. Boyko; Konstantin M. Polyakov; Dimitry Y. Sorokin; Renata A. Zvyagilskaya; Vladimir O. Popov (pp. 715-723).
A highly active cytochrome c nitrite reductase from the haloalkaliphilic sulfur-oxidizing non-ammonifying bacterium Tv. nitratireducens strain ALEN 2 (TvNiR) was isolated and purified to apparent electrophoretic homogeneity. The enzyme catalyzes reductive conversion of nitrite and hydroxylamine to ammonia without release of any intermediates, as well as reduction of sulfite to sulfide. TvNiR also possesses peroxidase activity. In solution TvNiR exists as a stable hexamer with molecular mass of about 360kDa. Each TvNiR subunit with molecular mass of 64kDa contains, as defined from spectral properties and sequence analysis, eight c-type haems. Seven of them are coordinated by the characteristic CXXCH motifs for haem c binding, while one is bonded by the unique CXXCK motif. So far, this motif coordinating the catalytic haem was found only in bacterial cytochrome c nitrite reductases (ccNiRs). All the residues essential for catalysis in the known ccNiRs were also identified in TvNiR. However, TvNiR is only distantly related to known bacterial ammonifying dissimilatory ccNiRs, sharing no more than 20% homology.
Keywords: Abbreviations; ABTS; 2,2′-azino-di(3-ethylbenzthiazoline-6-sulfonic acid); HAO; hydroxylamine oxidoreductase; MV; methyl viologen; NiR; nitrite reductase; ccNiR; cytochrome; c; nitrite reductase; TvNiR; nitrite reductase from; Thioalkalivibrio nitratireducens Thioalkalivibrio nitratireducens; Cytochrome; c; nitrite reductase; Haem; c; Sulfite reductase activity; Peroxidase activity; Oligomeric structure; Gene sequence
Low resolution structure of the human α4 protein (IgBP1) and studies on the stability of α4 and of its yeast ortholog Tap42 by Juliana Helena Costa Smetana; Cristiano Luiz Pinto Oliveira; Willy Jablonka; Thelma Aguiar Pertinhez; Flavia Raquel Gonçalves Carneiro; Monica Montero-Lomeli; Iris Torriani; Nilson Ivo Tonin Zanchin (pp. 724-734).
The yeast Tap42 and mammalian α4 proteins belong to a highly conserved family of regulators of the type 2A phosphatases, which participate in the rapamycin-sensitive signaling pathway, connecting nutrient availability to cell growth. The mechanism of regulation involves binding of Tap42 to Sit4 and PPH21/22 in yeast and binding of α4 to the catalytic subunits of type 2A-related phosphatases PP2A, PP4 and PP6 in mammals. Both recombinant proteins undergo partial proteolysis, generating stable N-terminal fragments. The full-length proteins and α4 C-terminal deletion mutants at amino acids 222 (α4Δ222), 236 (α4Δ236) and 254 (α4Δ254) were expressed in E. coli. α4Δ254 undergoes proteolysis, producing a fragment similar to the one generated by full-length α4, whereas α4Δ222 and α4Δ236 are highly stable proteins. α4 and Tap42 show α-helical circular dichroism spectra, as do their respective N-terminal proteolysis resistant products. The cloned truncated proteins α4Δ222 and α4Δ236, however, possess a higher content of α-helix, indicating that the C-terminal region is less structured, which is consistent with its higher sensitivity to proteolysis. In spite of their higher secondary structure content, α4Δ222 and α4Δ236 showed thermal unfolding kinetics similar to the full-length α4. Based on small angle X-ray scattering (SAXS), the calculated radius of gyration for α4 and Tap42 were 41.2±0.8 Å and 42.8±0.7 Å and their maximum dimension ∼142 Å and ∼147 Å, respectively. The radii of gyration for α4Δ222 and α4Δ236 were 21.6±0.3 Å and 25.7±0.2 Å, respectively. Kratky plots show that all studied proteins show variable degree of compactness. Calculation of model structures based on SAXS data showed that α4Δ222 and α4Δ236 proteins have globular conformation, whereas α4 and Tap42 exhibit elongated shapes.
Keywords: Tap42 protein family; Rapamycin signaling pathway; Small angle X-ray scattering
Iron–sulfur cluster reconstitution of spinach chloroplast Rieske protein requires a partially prefolded apoprotein by Beata Gubernator; Jaroslaw Króliczewski; Toivo Kallas; Andrzej Szczepaniak (pp. 735-742).
The Rieske 2Fe–2S protein is a central component of the photosynthetic electron transport cytochrome b6 f complex in chloroplast and cyanobacterial thylakoid membranes. We have constructed plasmids for expression in Escherichia coli of full-length and truncated Spinacia oleracea Rieske (PetC) proteins fused to the MalE, maltose binding protein. The expressed Rieske fusion proteins were found predominantly in soluble form in the E. coli cytoplasm. These proteins could be readily purified for further experimentation. In vitro reconstitution of the characteristic, “Rieske-type� 2Fe–2S cluster into these fused proteins was accomplished by a chemical method employing reduced iron and sulfide. Cluster incorporation was monitored by electron paramagnetic resonance and optical circular dichroism (CD) spectroscopy. CD spectral analysis in the ultraviolet region suggests that the spinach Rieske apoprotein must be in a partially folded conformation to incorporate an appropriate iron–sulfur cluster. These data further suggest that upon cluster integration, further folding occurs, allowing the Rieske protein to attain a final, native structure. The data presented here are the first to demonstrate successful chemical reconstitution of the 2Fe–2S cluster into a Rieske apoprotein from higher plant chloroplasts.
Keywords: Rieske protein; Protein folding; 2Fe–2S center; Metallo-cluster reconstitution; Cytochrome; b; 6; f; complex
Oxidation of Arg-410 promotes the elimination of human serum albumin by Yasunori Iwao; Makoto Anraku; Keishi Yamasaki; Ulrich Kragh-Hansen; Keiichi Kawai; Toru Maruyama; Masaki Otagiri (pp. 743-749).
The effect of the oxidation of amino acid residues on albumin on its in vivo elimination was investigated using mutants and oxidized HSAs. The single-residue mutants (H146A, K199A, W214A, R218H, R410A, Y411A) and oxidized HSAs were produced by the recombinant DNA techniques and incubation with a metal ion-catalyzed oxidation (MCO) system for 12, 24, 48 or 72 h. Pharmacokinetics were evaluated in mice after labeling with111In. Structural and functional properties were examined by several spectroscopic techniques. Time-dependent increase in carbonyl group content resulted in increase in the liver clearance of oxidized HSAs. Slight decreases in α-helical content as the result of oxidation was induced by the increases in accessible hydrophobic areas and the net negative charge on the HSA molecule. No significant change in the pharmacokinetics and structural properties was observed for the W214A, R218H and Y411A mutants, but the properties for the H146A, K199A and R410A mutants were affected (extent of effect: R410A>K199A>H146A). The liver clearance of these proteins is closely correlated to hydrophobicity ( r=0.929, P<0.01) and the net charge of the proteins ( r=0.930, P<0.01). The rate of elimination of HSA is closely related to the hydrophobicity and net charge of the molecule. Further, the R410A mutants had a short half-life and structure similar to oxidized HSA after oxidation. Therefore, the modification of Arg-410 via oxidative stress may promote the elimination of HSA.
Keywords: Abbreviations; HSA; human serum albumin; rHSA; recombinant HSA; MCO; 12; -HSA, MCO; 24; -HSA, MCO; 48; -HSA and MCO; 72; -HSA; HSA oxidized by metal ion-catalysis for 12, 24, 48 and 72 h, respectively; bis-ANS; 1,1-bis-4-anilino-naphthalene-5,5-sulfonic acidHuman serum albumin; Oxidation; Arg-410; Elimination; Liver clearance
Tryptophanase from Proteus vulgaris: The conformational rearrangement in the active site, induced by the mutation of Tyrosine 72 to Phenylalanine, and its mechanistic consequences by Vitalia V. Kulikova; Ludmila N. Zakomirdina; Irene S. Dementieva; Robert S. Phillips; Paul D. Gollnick; Tatyana V. Demidkina; Nicolai G. Faleev (pp. 750-757).
Tyr72 is located at the active site of tryptophanase (Trpase) from Proteus vulgaris. For the wild-type Trpase Tyr72 might be considered as the general acid catalyst at the stage of elimination of the leaving groups. The replacement of Tyr72 by Phe leads to a decrease in activity forl-tryptophan by 50,000-fold and to a considerable rearrangement of the active site of Trpase. This rearrangement leads to an increase of room around the α-C atom of any bound amino acid, such that covalent binding of α-methyl-substituted amino acids becomes possible (which cannot be realized in wild-type Trpase). The changes in reactivities of S-alkyl-l-cysteines provide evidence for an increase of congestion in the proximity of their side groups in the mutant enzyme as compared to wild-type enzyme. The observed alteration of catalytic properties in a large degree originates from a conformational change in the active site. The Y72F Trpase retains significant activity forl-serine, which allowed us to conclude that in the mutant enzyme, some functional group is present which fulfills the role of the general acid catalyst in reactions associated with elimination of small leaving groups.
Keywords: Proteus vulgaris; Pyridoxal 5′-phosphate; Site-directed mutagenesis; Tryptophan indole-lyase
Hemoglobin I from Lucina pectinata: A model for distal heme-ligand control by Ruth Pietri; Ruth G. León; Laurent Kiger? ??; Michael C. Marden; Laura B. Granell; Carmen L. Cadilla; Juan López-Garriga (pp. 758-765).
Lucina pectinata hemoglobin I (HbI), which is a ferric sulfide-reactive hemeprotein, contains a distal pocket characterized by the presence of GlnE7 and PheB10. To elucidate the structural-functional properties of HbI, oxygen binding kinetics and FTIR studies with recombinant HbI (rHbI) and a set of mutants were conducted using CO and CN− as sensors of the hemeprotein environment. Three νCO modes were observed for rHbI at 1936 cm−1 ( A3, closed conformer) 1950 cm−1 ( A1,2, closed conformer) and 1960 cm−1( A0, open conformer). These νCO were affected by substitution of GlnE7 and PheB10 in the CO complexes. The contribution of GlnE7 is demonstrated when this residue is replaced with Asn, Val or His. For instance, decreasing the positive electrostatic environment with GlnE7Val, causes an increase of 65% in the population of A0 and the disappearance and 55% reduction of the population of the A1,2 and A3 respectively. The contribution of PheB10 to the stabilization of ligands is also observed in the Leu and Tyr mutants. The PheB10Leu mutation produced an 8% decrease in the population of the A3 conformer while that of the A1,2 configuration increased by 30%. This suggests that GlnE7 and PheB10 contribute to the A3 conformer stabilizing the CO in a closed configuration. With CN− as probe no substantial differences in the νCN was observed upon substitution of GlnE7 by Val while a slight down shift in the νCN from 2120 cm−1 to 2117 cm−1 was observed in the PheB10Leu mutant. This implies that in HbICN GlnE7 moves away from the binding site while PheB10 remains in the vicinity of the bound CN−. Here, a mechanism in which the flexibility of the distal protein matrix coupled with hemeporphyrin movement toward a different configuration is suggested as an important process in the H2S transport and delivery in hemoglobin I.
Keywords: Hemoglobin I; Fourier transform infrared resonance; Recombinant HbI; Carbon monoxide; Cyanide; Hydrogen sulfide
Prediction of the binding mode between BMS-378806 and HIV-1 gp120 by docking and molecular dynamics simulation by Ren Kong; Jian Jun Tan; Xiao Hui Ma; Wei Zu Chen; Cun Xin Wang (pp. 766-772).
BMS-378806 is a newly discovered small molecule that effectively blocks the binding of CD4 with gp120. The binding mode of this kind of inhibitor remains unknown. In this paper, AutoDock 3.0 in conjunction with molecular dynamics simulation, accommodating the receptor's flexibility, was used to explore the binding mode between BMS-378806 and gp120. Two structures, Mode I and Mode II, with the lowest docking energy were selected as different representative binding modes. The analysis of the results from the molecular dynamics simulation indicated that the binding of BMS-348806 in Mode II is more stable. The average structure of Mode II was analyzed and compared with the experimental data. The conclusion was that BMS-378806 inserts the azaindole ring deeply into the PHE43 cavity and makes contact with a number of residues in the cavity, on the cavity and near the cavity. This study benefits the understanding of the mechanism of this kind of inhibitor and may provide useful information for rational drug design.
Keywords: Abbreviations; MD; molecular dynamics; RMSD; root mean square deviationBMS-378806; HIV-1; gp120; Docking; Molecular Dynamics
Reduced expression of regucalcin in young and aged mdx diaphragm indicates abnormal cytosolic calcium handling in dystrophin-deficient muscle by Philip Doran; Paul Dowling; Pamela Donoghue; Maria Buffini; Kay Ohlendieck (pp. 773-785).
The cytosolic Ca2+-binding protein regucalcin is involved in intracellular signaling and present in high abundance in the liver. Here, we could show by comparative mass spectrometry-based proteomics screening of normal versus dystrophic fibres that regucalcin of 33.9 kDa and pI5.2 also exists in diaphragm muscle. Since the expression of sarcolemmal Ca2+-leak channels and luminal Ca2+-binding elements is altered in dystrophin-deficient muscle, we initiated this study in order to determine whether additional soluble muscle proteins involved in Ca2+-handling are affected in muscular dystrophy. Following separation by two-dimensional gel electrophoresis, the spot pattern of the normal versus the mdx diaphragm muscle proteome was evaluated by densitometry. The expression levels of 20 major protein spots were shown to change and their identity determined by mass spectrometry. A 2-fold reduction of regucalcin in mdx diaphragm, as well as in dystrophic limb muscle and heart, was confirmed by immunoblotting in both young and aged mdx mice. The results from our proteomics analysis of dystrophic diaphragm support the concept that abnormal Ca2+-handling is involved in x-linked muscular dystrophy. The reduction in key Ca2+-handling proteins may result in an insufficient maintenance of Ca2+-homeostasis and an abnormal regulation of Ca2+-dependent enzymes resulting in disturbed intracellular signaling mechanisms in dystrophinopathies.
Keywords: mdx diaphragm; Calcium homeostasis; Regucalcin; Muscle proteomics; Muscular dystrophy
Kinetics of regulatory serine variants of tyrosine hydroxylase with cyclic AMP-dependent protein kinase and extracellular signal-regulated protein kinase 2 by Montserrat Royo; S. Colette Daubner (pp. 786-792).
Rat tyrosine hydroxylase is phosphorylated at four serine residues, at positions 8, 19, 31, and 40 in its amino terminal regulatory domain by multiple protein kinases. Cyclic AMP-dependent protein kinase phosphorylates S40, which results in alleviation of inhibition by dopamine. Extracellular signal-regulated protein kinase 2 phosphorylates S8 and S31. Site-directed serine-to-glutamate mutations were introduced into tyrosine hydroxylase to mimic prior phosphorylation of the regulatory serines; these proteins were used as substrates for cAMP-dependent kinase and extracellular signal-regulated kinase 2. The activity of cAMP-dependent kinase was unaffected by the substitution of serines 8, 19 or 31 with glutamate and the activity of extracellular signal-regulated kinase 2 was unaffected by substitution of serines 19 or 40 with glutamate. Cyclic AMP-dependent kinase was less active in phosphorylating S40 if dopamine was bound to tyrosine hydroxylase, but extracellular signal-regulated kinase 2 phosphorylation at S31 was unaffected by the presence of dopamine.
Keywords: Abbreviations; TyrH; tyrosine hydroxylase; PKA; cAMP-dependent protein kinase; CaMKII; Ca; 2+; /calmodulin-dependent protein kinase; ERK2; extracellular signal-regulated kinase 2; MAPKAPK-2; mitogen-activated-protein-kinase-activated protein-kinase 2; Cdk5; cyclin-dependent kinase 5; TCA; trichloroacetic acid; TyrHpS8; tyrosine hydroxylase phosphorylated at S8; TyrHpS19; tyrosine hydroxylase phosphorylated at S19; TyrHpS31; tyrosine hydroxylase phosphorylated at S31; TyrHpS40; tyrosine hydroxylase phosphorylated at S40; R domains; regulatory domains,PH; 4; , tetrahydropterin; PH; 3; OH; C4a-hydroxypterinTyrosine hydroxylase; cAMP-dependent protein kinase; Extracellular signal-regulated kinase 2 Protein phosphorylation; Feedback inhibition; Catecholamine synthesis
Structural analysis of protein inclusion bodies by Fourier transform infrared microspectroscopy by Diletta Ami; Antonino Natalello; Geoffrey Taylor; Giancarlo Tonon; Silvia Maria Doglia (pp. 793-799).
The expression of recombinant human growth hormone (h-GH) and human interferon-alpha-2b (IFN-alpha-2b) in E. coli leads to the formation of insoluble protein aggregates or inclusion bodies (IBs). The secondary structure of these IBs, their corresponding native forms and thermal aggregates were studied by Fourier Transform Infrared (FT-IR) spectroscopy and microspectroscopy. It was demonstrated that residual native-like structures were maintained within IBs at different extents depending on the level of expression, with possible implications in biotechnology. Furthermore, comparison between infrared spectra of thermal aggregates and IBs suggests new insights on the structure of protein aggregates.
Keywords: Protein aggregation; Inclusion bodies; Heterologous protein production; FT-IR spectroscopy; Human growth hormone; Interferon alpha
Kinetic study of racemization of aspartyl residues in recombinant human αA-crystallin by Tooru Nakamura; Yutaka Sadakane; Noriko Fujii (pp. 800-806).
Asp58 and Asp151 in human lens αA-crystallin invert and isomerize tod-β-aspartyl residues with age. Here, we report that the racemization rate constants ( k) of Asp58 and Asp151 residues in human recombinant αA-crystallin at 37 °C are 3.72±0.8×10−4 and 10.7±0.7×10−4/day, respectively. The activation energy of racemization of Asp58 and Asp151 in the protein was 27.0±0.5 kcal/mol and 21.0±0.5 kcal/mol, respectively. The time required for the D/ L ratio of Asp58 and Asp151 to approximate to 1.0 ( D/ L ratio of Asp=0.99) at 37 °C was estimated as 20.9±3.7 and 6.80±0.4 years, respectively. Thus, Asp151 is more susceptible to racemization than Asp58, consistent with data from short model peptides. However, the racemization rates of both Asp58 and Asp151 residues in the protein were twice as rapid as in model peptides. These results indicate that the racemization of Asp residues in αA-crystallin may be influenced not only by the primary structure but also by the higher order structure around Asp residues in the protein.
Keywords: d; -aspartyl residue; Kinetics; Lens; Racemization; Recombinant αA-crystallin
Fluorescence properties and conformational stability of the β-hemocyanin of Helix pomatia by Krassimira Idakieva; Nurul I. Siddiqui; Katja Parvanova; Peter Nikolov; Constant Gielens (pp. 807-814).
The β-hemocyanin (β-HpH) is one of the three dioxygen-binding proteins found freely dissolved in the hemolymph of the gastropodan mollusc Helix pomatia. The didecameric molecule (molecular mass 9 MDa) is built up of only one type of subunits. The fluorescence properties of the oxygenated and apo-form (copper-deprived) of the didecamer and its subunits were characterized. Upon excitation of the hemocyanins at 295 or 280 nm, tryptophyl residues buried in the hydrophobic interior of the protein determine the fluorescence emission. This is confirmed by quenching experiments with acrylamide, cesium chloride and potassium iodide. The copper–dioxygen system at the binuclear active site quenches the tryptophan emission of the oxy-β-HpH. The removal of this system increases the fluorescence quantum yield and causes structural rearrangement of the microenvironment of the emitting tryptophyl residues in the apo-form. Time-resolved fluorescence measurements show that the oxygenated and copper-deprived forms of the β-HpH and its subunits exist in different conformations. The thermal stability of the oxy- and apo-β-HpH is characterized by a transition temperature ( Tm) of 84 °C and 63 °C, respectively, obtained by differential scanning calorimetry. Increase of the temperature influences the active site at lower temperatures than the environments of tryptophans and tyrosines causing a loss of oxygen bound to the copper atoms. This process is, at least partially, reversible as after cooling of the protein samples, around 60% reinstatement of the copper-peroxide band has been observed. The results confirm the role of the copper–dioxygen complex for the stabilization of the hemocyanin structure in solution. The other important stabilizing factor is oligomerization of the hemocyanin molecule.
Keywords: Abbreviations; DSC; differential scanning calorimetry; EDTA; ethylenediaminetetraacetic acid; Hc; hemocyanin; HpH; Helix pomatia; hemocyanin; MOPS; 3-[; N; -Morpholino]-propanesulfonic acid; Ac-Trp-NH; 2; N; -acetyltryptophanamide; Tris/HCl; tris (hydroxymethyl) amino-methane hydrochlorideHemocyanin; Mollusca; Helix pomatia; Fluorescence spectroscopy; Differential scanning calorimetry
The role of β2-glycoprotein I (β2GPI) in the activation of plasminogen by Francisco López-Lira; Luis Rosales-León; Verónica Monroy MartÃnez; Blanca H. Ruiz Ordaz (pp. 815-823).
β2-glycoprotein I (β2GPI) is a glycoprotein of unknown physiological function. It is the main target antigen for antiphospholipid antibodies in patients with antiphospholipid syndrome (APS). β2GPI binds with high affinity to the atherogenic lipoprotein Lp(a) which shares structural homology with plasminogen, a key molecule in the fibrinolytic system. Impaired fibrinolysis has been described in APS. The present work reports the interaction between β2GPI and Glu-Plasminogen which may explain the recently described proteolytic effect of plasmin on β2GPI. In the process of Glu-Plasminogen activation, we found an increase in plasmin generation both at fibrin and cellular surface level as a function of the concentration of β2GPI added, suggesting an important role as a cofactor in the trimolecular complex β2GPI-Plasminogen-tPA. This phenomenon represents a novel regulatory step both in the positive feedback mechanism for extrinsic fibrinolysis and in antithrombotic regulation. IgG anti-β2GPI antibodies recognized the β2GPI at the endothelial surface inducing its activation with an increase of ICAM-I and a decrease in the expression of thrombomodulin favoring a pro-thrombotic state in the vascular endothelium. The interference in the plasmin conversion by anti-β2GPI antibodies could generate thrombosis as observed in APS.
Keywords: β2-glycoprotein; Plasmin; Plasminogen activation; Fibrinolysis; Thrombosis
The role of tyrosine 177 in human 11β-hydroxysteroid dehydrogenase type 1 in substrate and inhibitor binding: an unlikely hydrogen bond donor for the substrate by Ki Won Kim; Zhulun Wang; James Busby; Trace Tsuruda; Michelle Chen; Clarence Hale; VÃctor M. Castro; Stefan Svensson; Rebecca Nybo; Fei Xiong; Minghan Wang (pp. 824-830).
The catalytic motif (YSASK) at the active site of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is conserved across different species. The crystal structures of the human, guinea pig and mouse enzymes have been resolved to help identify the non-conserved residues at the active site. A tyrosine residue (Y177) upstream of the catalytic motif in human 11β-HSD1 represents the largest difference at the active sites between the human and the rodent enzyme where the corresponding residue is glutamine. Although Y177 was postulated as a potential hydrogen bond donor in substrate binding in crystal structure-based modeling, no experimental evidence is available to support this notion. Here, we report that Y177 is not a hydrogen bond donor in substrate binding because removal of the hydroxyl group from its side chain by mutagenesis (Y177F) did not significantly change the Km value for cortisone. However, removal of the hydrophobic side chain by changing tyrosine to alanine (Y177A) or substitution with a hydrophilic side chain by changing tyrosine to glutamine (Y177Q) increased Km values for cortisone. These data suggest that Y177 is involved in substrate binding through its hydrophobic side chain but not by hydrogen bonding. In addition, the three mutations had little effect on the binding of the rodent substrate 11-dehydrocorticosterone, suggesting that Y177 does not confer substrate specificity. However, the same mutations reduced the affinity of the licorice derived 11β-HSD1 inhibitor glycyrrhetinic acid by about 6- to 10-fold. Interestingly, the affinity of carbenoxolone, the hemisuccinate ester of glycyrrhetinic acid with a similar potency against the wildtype enzyme, was not drastically affected by the same mutations at Y177. These data suggest that Y177 has a unique role in inhibitor binding. Molecular modeling with glycyrrhetinic acid led to findings consistent with the experimental data and provided potential interaction mechanisms. Our data suggest that Y177 plays an important role in both substrate and inhibitor binding but it is unlikely a hydrogen bond donor for the substrate.
Keywords: Abbreviations; CBX; carbenoxolone; ER; endoplasmic reticulum; GE; glycyrrhetinic acid; GR; glucocorticoid receptor; 11β-HSD1; 11β-hydroxysteriod dehydrogenase type 1; 11-DHC; 11-dehydrocorticosterone; SCAD or SDR; short chain alcohol dehydrogenase or short chain dehydroganase/reductase11β-hydroxysteroid dehydrogenase; GR; glucocorticoid receptor; Cortisone; Carbenoxolone; Site-directed mutagenesis
Unfolded, oxidized, and thermoinactivated forms of glyceraldehyde-3-phosphate dehydrogenase interact with the chaperonin GroEL in different ways by I.N. Naletova; V.I. Muronetz; E.V. Schmalhausen (pp. 831-838).
The interaction of GroEL with different denatured forms of glyceraldehyde-3-phosphate dehydrogenase* (GAPDH) has been investigated. GroEL does not prevent thermal denaturation of GAPDH, but effectively interacts with the thermodenatured enzyme, thus preventing the aggregation of denatured molecules. Binding of the thermodenatured GAPDH shifts the Tm value of the GroEL thermodenaturation curve by 3° towards higher temperatures and increases the Δ Hcal value 1.44-fold, indicating a significant increase in the thermal stability of the resulting complex. GAPDH thermodenatured in the presence of GroEL cannot be reactivated by the addition of GroES, Mg2+, and ATP. In contrast, GAPDH denatured in guanidine hydrochloride (GAPDHden) is reactivated in the presence of GroEL, GroES, Mg2+, and ATP, yielding 11–15% of its original activity, while the spontaneous reactivation yields only 2–3%. The oxidation of GAPDH with hydrogen peroxide in the presence of 4 M guanidine hydrochloride results in the formation of the enzyme (GAPDHox) that cannot acquire its native conformation and binds to GroEL irreversibly. Binding of GAPDHox to one of the GroEL rings completely inhibits the GroEL-assisted reactivation of GAPDHden, but does not affect the GroEL-assisted reactivation of lactate dehydrogenase (LDH). The data suggest that LDH can be successfully reactivated due to the binding of the denatured molecules to the apical domain of the opposite GroEL ring with their subsequent release into the solution without encapsulation ( trans-mechanism). In contrast, GAPDH requires the hydrophilic cavity for the reactivation ( cis-mechanism).
Keywords: Abbreviations; GAPDH, glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12); GAPDH; ox; , GAPDH oxidized with H; 2; O; 2; in the presence of guanidine hydrochloride; GAPDH; den; , GAPDH denatured in guanidine hydrochloride; LDH, lactate dehydrogenase (EC 1.1.1.27)Glyceraldehyde-3-phosphate dehydrogenase; GroEL; Oxidation; Protein folding
Purification and preliminary X-ray crystallographic analysis of the ligand-binding domain of Sinorhizobium meliloti DctB by Beiyan Nan; Yanfeng Zhou; Yu-He Liang; Jin Wen; Qi Ma; Siwei Zhang; Yiping Wang; Xiao-Dong Su (pp. 839-841).
Sinorhizobium meliloti DctBD is a well-characterized two-component system. It is believed that DctB senses the concentration of C4-dicarboxylate compounds on the outside of the bacterium and phosphorylates DctD, which in turn activates transcription of the dctA gene, coding for a gene of C4-dicarboxylate permease. The structure and function of the ligand-binding domain of DctB has not been thoroughly investigated. In this study, this domain was produced in E. coli in soluble form, and purified to homogeneity. Crystals were obtained by hanging-drop vapor-diffusion method. The crystals diffracted to 2.3 Å resolution and belonged to P42 space group with unit cell dimensions of a = b = 71.77 Å, c = 227.14 Å. The asymmetric unit contains four molecules with a corresponding VM of 2.4 Å3 Da−1 and a solvent content of 49.1%.
Keywords: Sinorhizobium meliloti; DctB; Ligand-binding domain; Protein crystallization; X-ray diffraction
Crystallisation and preliminary structure determination of a NADH: quinone oxidoreductase from the extremophile Acidianus ambivalens by José A. Brito; Tiago M. Bandeiras; Miguel Teixeira; Clemens Vonrhein; Margarida Archer (pp. 842-845).
NADH:quinone oxidoreductases (NDHs), constitute one of the electron entry points into membrane-bound respiratory chains, oxidising NADH and reducing quinones. Type-II NDHs (NDH-2) are functionally unable to translocate protons and are typically constituted by a single ∼50 kDa subunit lacking iron–sulfur clusters and containing one flavin as the sole redox centre. No three dimensional crystal structure is yet available for NDHs. We describe the crystallisation and preliminary structure determination of a NDH-2 that contains a covalently bound FAD, isolated from the membrane fraction of Acidianus ambivalens, a hyperthermoacidophilic archaeon capable of growing at 80 °C and pH 2.0. NDH-2 was solubilised with the detergent n-dodecyl-β-d-maltoside and crystallised using ammonium phosphate as precipitant. The structure was solved by MIRAS using Pt and I derivatives.
Keywords: Crystallisation; NADH:quinone oxidoreductase; Membrane protein; Aerobic respiratory chain; Extremophile; Archaea