Bound Form (bound + form)

Distribution by Scientific Domains

Selected Abstracts

Enzymatic and immunochemical evaluation of phospholipid hydroperoxide glutathione peroxidase (PHGPx) in testes and epididymal spermatozoa of rats of different ages

Federica Tramer
Selenium (Se) and selenoproteins such as glutathione peroxidases are necessary for the proper development and fertilizing capacity of sperm cells. Phospholipid hydroperoxide glutathione peroxidase (PHGPx, E.C. is a monomeric seleno-enzyme present in different mammalian tissues in soluble and bound form. Its function, like the other glutathione peroxidases, was originally viewed as a protective role against hydroperoxides, but direct and indirect evidence indicates that it has additional regulatory roles. PHGPx is present in testis cells and sperm cells, and its appearance is hormone regulated. We present here biochemical data, which clearly indicate that the enzyme specific activity in rat is age-dependent during the life-span monitored (from 36 to 365 days), with a maximum at 3 months of age in the testis germ cells and at 6 months of age in the isolated epididymal sperm cells. Western blotting and immunocytochemical analysis by means of anti-PHGPx antibodies show the different distribution and the strong binding of PHGPx in the testes and sperm cell subcellular compartments (nucleus, acrosome, mitochondria and residual bodies) of rats of different age. The presence of the protein exhibits in the testis cells a pattern different from that of the catalytic activity, with a maximum at 6 months of age. The subcellular distribution of PHGPx is qualitatively, but not quantitatively, unchanged during ageing. These different behaviours are compared and discussed. [source]

Insights on protein-DNA recognition by coarse grain modelling

P. Poulain
Abstract Coarse grain modelling of macromolecules is a new approach, potentially well adapted to answer numerous issues, ranging from physics to biology. We propose here an original DNA coarse grain model specifically dedicated to protein-DNA docking, a crucial, but still largely unresolved, question in molecular biology. Using a representative set of protein-DNA complexes, we first show that our model is able to predict the interaction surface between the macromolecular partners taken in their bound form. In a second part, the impact of the DNA sequence and electrostatics, together with the DNA and protein conformations on docking is investigated. Our results strongly suggest that the overall DNA structure mainly contributes in discriminating the interaction site on cognate proteins. Direct electrostatic interactions between phosphate groups and amino acid side chains strengthen the binding. Overall, this work demonstrates that coarse grain modeling can reveal itself a precious auxiliary for a general and complete description and understanding of protein-DNA association mechanisms. 2008 Wiley Periodicals, Inc. J Comput Chem, 2008 [source]

Detection of cometary amines in samples returned by Stardust

Daniel P. Glavin
A suite of amino acids and amines including glycine, L-alanine, ,-alanine (BALA), ,-amino- n -butyric acid (GABA), ,-amino- n -caproic acid (EACA), ethanolamine (MEA), methylamine (MA), and ethylamine (EA) were identified in acid-hydrolyzed, hot-water extracts of these Stardust materials above background levels. With the exception of MA and EA, all other primary amines detected in comet-exposed aerogel fragments C2054,4 and C2086,1 were also present in the flight aerogel witness tile that was not exposed to the comet, indicating that most amines are terrestrial in origin. The enhanced relative abundances of MA and EA in comet-exposed aerogel compared to controls, coupled with MA to EA ratios (C2054,4: 1.0 0.2; C2086,1: 1.8 0.2) that are distinct from preflight aerogels (E243,13C and E243,13F: 7 3), suggest that these volatile amines were captured from comet Wild 2. MA and EA were present predominantly in an acid-hydrolyzable bound form in the aerogel, rather than as free primary amines, which is consistent with laboratory analyses of cometary ice analog materials. It is possible that Wild 2 MA and EA were formed on energetically processed icy grains containing ammonia and approximately equal abundances of methane and ethane. The presence of cometary amines in Stardust material supports the hypothesis that comets were an important source of prebiotic organic carbon and nitrogen on the early Earth. [source]

IscR acts as an activator in response to oxidative stress for the suf operon encoding Fe-S assembly proteins

Won-Sik Yeo
Summary In Escherichia coli, Fe-S clusters are assembled by gene products encoded from the isc and suf operons. Both the iscRSUA and sufABCDSE operons are induced highly by oxidants, reflecting an increased need for providing and maintaining Fe-S clusters under oxidative stress conditions. Three cis -acting oxidant-responsive elements (ORE-I, II, III) in the upstream of the sufA promoter serve as the binding sites for OxyR, IHF and an uncharacterized factor respectively. Using DNA affinity fractionation, we isolated an ORE-III-binding factor that positively regulates the suf operon in response to various oxidants. MALDI-TOF mass analysis identified it with IscR, known to serve as a repressor of the iscRSUA gene expression under anaerobic condition as a [2Fe-2S]-bound form. The iscR null mutation abolished ORE-III-binding activity in cell extracts, and caused a significant decrease in the oxidant induction of sufA in vivo. OxyR and IscR contributed almost equally to activate the sufA operon in response to oxidants. Purified IscR that lacked Fe-S cluster bound to the ORE-III site and activated transcription from the sufA promoter in vitro. Mutations in Fe-S-binding sites of IscR enabled sufA activation in vivo and in vitro. These results support a model that IscR in its demetallated form directly activates sufA transcription, while it de-represses isc operon, under oxidative stress condition. [source]

Amino Acid Residues in GRK1/GRK7 Responsible for Interaction with S-Modulin/Recoverin,

Aya Torisawa
GRK1 is a visual pigment kinase in rods and is essential for inactivation of light-activated rhodopsin. The GRK1 activity is inhibited by binding of the Ca2+ -bound form of S-modulin/recoverin. We previously identified the S-modulin/recoverin site to interact with GRK1. In the present study, we identified its counterpart in GRK1. We synthesized 29 of GRK1 or GRK7 partial peptides that cover the entire sequence of GRK1/GRK7, and examined whether these peptides inhibit S-modulin/recoverin activity most probably by preoccupying the binding site for GRK1. The inhibition was the greatest with the N-terminal peptide (p1, aa 3,23 in GRK7). On mutation of each of eight amino acid residues highly conserved in the p1 region of more than 10 orthologs, the inhibition was significantly reduced in the mutation of Leu6, Asn12 and Tyr15. We further examined the binding of the peptides, including mutated ones, to S-modulin/recoverin with a resonance mirror biosensor. The binding correlated well with the degree of the inhibition by a peptide. The inhibition, therefore, seemed to be due to a direct binding of the kinase peptide to the binding site of active S-modulin/recoverin. A GRK1 region close to its C-terminus also seemed to be the binding site for S-modulin/recoverin. [source]

Protein,protein docking with multiple residue conformations and residue substitutions

David M. Lorber
Abstract The protein docking problem has two major aspects: sampling conformations and orientations, and scoring them for fit. To investigate the extent to which the protein docking problem may be attributed to the sampling of ligand side-chain conformations, multiple conformations of multiple residues were calculated for the uncomplexed (unbound) structures of protein ligands. These ligand conformations were docked into both the complexed (bound) and unbound conformations of the cognate receptors, and their energies were evaluated using an atomistic potential function. The following questions were considered: (1) does the ensemble of precalculated ligand conformations contain a structure similar to the bound form of the ligand? (2) Can the large number of conformations that are calculated be efficiently docked into the receptors? (3) Can near-native complexes be distinguished from non-native complexes? Results from seven test systems suggest that the precalculated ensembles do include side-chain conformations similar to those adopted in the experimental complexes. By assuming additivity among the side chains, the ensemble can be docked in less than 12 h on a desktop computer. These multiconformer dockings produce near-native complexes and also non-native complexes. When docked against the bound conformations of the receptors, the near-native complexes of the unbound ligand were always distinguishable from the non-native complexes. When docked against the unbound conformations of the receptors, the near-native dockings could usually, but not always, be distinguished from the non-native complexes. In every case, docking the unbound ligands with flexible side chains led to better energies and a better distinction between near-native and non-native fits. An extension of this algorithm allowed for docking multiple residue substitutions (mutants) in addition to multiple conformations. The rankings of the docked mutant proteins correlated with experimental binding affinities. These results suggest that sampling multiple residue conformations and residue substitutions of the unbound ligand contributes to, but does not fully provide, a solution to the protein docking problem. Conformational sampling allows a classical atomistic scoring function to be used; such a function may contribute to better selectivity between near-native and non-native complexes. Allowing for receptor flexibility may further extend these results. [source]

Functional role of Coenzyme Q in the energy coupling of NADH-CoQ oxidoreductase (Complex I): Stabilization of the semiquinone state with the application of inside-positive membrane potential to proteoliposomes

BIOFACTORS, Issue 1-4 2008
Tomoko Ohnishi Ph.D.
Abstract Coenzyme Q10 (which is also designated as CoQ10, ubiquinone-10, UQ10, CoQ, UQ or simply as Q) plays an important role in energy metabolism. For NADH-Q oxidoreductase (complex I), Ohnishi and Salerno proposed a hypothesis that the proton pump is operated by the redox-driven conformational change of a Q-binding protein, and that the bound form of semiquinone (SQ) serves as its gate [FEBS Letters 579 (2005) 45,55]. This was based on the following experimental results: (i) EPR signals of the fast-relaxing SQ anion (designated as Q) are observable only in the presence of the proton electrochemical potential (,,); (ii) iron-sulfur cluster N2 and Q are directly spin-coupled; and (iii) their center-to-center distance was calculated as 12, but Q is only 5 deeper than N2 perpendicularly to the membrane. After the priming reduction of Q to Nf, the proton pump operates only in the steps between the semiquinone anion (Q) and fully reduced quinone (QH2). Thus, by cycling twice for one NADH molecule, the pump transports 4H+ per 2e,. This hypothesis predicts the following phenomena: (a) Coupled with the piericidin A sensitive NADH-DBQ or Q1 reductase reaction, ,, would be established; (b) ,, would enhance the SQ EPR signals; and (c) the dissipation of ,, with the addition of an uncoupler would increase the rate of NADH oxidation and decrease the SQ signals. We reconstituted bovine heart complex I, which was prepared at Yoshikawa's laboratory, into proteoliposomes. Using this system, we succeeded in demonstrating that all of these phenomena actually took place. We believe that these results strongly support our hypothesis. [source]

Crystallization and X-ray diffraction analysis of N-terminally truncated human ALG-2

Hironori Suzuki
ALG-2 (apoptosis-linked gene 2) is an apoptosis-linked calcium-binding protein with five EF-hand motifs in the C-terminal region. N-terminally truncated ALG-2 (des3-23ALG-2) was crystallized by the vapour-diffusion method in buffer consisting of either 50,mM MES pH 6.5, 12.5%(v/v) 2-propanol and 150,mM calcium acetate or 100,mM MES pH 6.0, 15%(v/v) ethanol and 200,mM zinc acetate. Crystals of the Ca2+ -bound form belonged to space group P212121, with unit-cell parameters a = 54.8, b = 154.4, c = 237.7,, , = , = , = 90, and diffracted to 3.1, resolution. Crystals of the Zn2+ -bound form belonged to space group P212121, with unit-cell parameters a = 52.8, b = 147.5, c = 230.7,, , = , = , = 90, and diffracted to 3.3, resolution. The structures of the Ca2+ -bound form and the Zn2+ -bound form were solved by the molecular-replacement method. Although both crystals contained eight ALG-2 molecules per asymmetric unit, the metal-ion locations and octameric arrangements were found to be significantly different. [source]

New insights into the binding mode of coenzymes: structure of Thermus thermophilus,1 -pyrroline-5-carboxylate dehydrogenase complexed with NADP+

Eiji Inagaki
,1 -Pyrroline-5-carboxylate dehydrogenase (P5CDh) is known to preferentially use NAD+ as a coenzyme. The kcat value of Thermus thermophilus P5CDh (TtP5CDh) is four times lower for NADP+ than for NAD+. The crystal structure of NADP+ -bound TtP5CDh was solved in order to study the structure,activity relationships for the coenzymes. The binding mode of NADP+ is essentially identical to that in the previously solved NAD+ -bound form, except for the regions around the additional 2,-phosphate group of NADP+. The coenzyme-binding site can only accommodate this group by the rotation of a glutamate residue and subtle shifts in the main chain. The 2,-phosphate of NADP+ increases the number of hydrogen bonds between TtP5CDh and NADP+ compared with that between TtP5CDh and NAD+. Furthermore, the phosphate of the bound NADP+ would restrict the `bending' of the coenzyme because of steric hindrance. Such bending is important for dissociation of the coenzymes. These results provide a plausible explanation of the lower turnover rate of NADP+ compared with NAD+. [source]

Effect of fermentation on free and bound volatile compounds of orange juice

Gang Fan
Abstract Aroma is one of the most important attributes of orange wine quality. The volatile compounds in orange wine mainly derive from oranges, yeast fermentation and compounds released from odourless glycosidic precursors present in the orange. In this study, free volatile compounds in orange juice and wine made from Citrus sinensis (L). Osbeck cv. Washington Sanguine were analysed by SPME-GC,MS. Bound fractions were isolated and extracted with methanol and Amberlite XAD-2 resin and then hydrolysed by almond , -glucosidase. Totals of 31 and 19 free volatiles were identified in orange juice and wines, respectively. Terpenes were the most abundant compounds in orange juice, while esters were quantitatively the dominant group in orange wine and most of them were compounds newly formed during fermentation, such as isoamyl acetate, ethyl hexanoate, ethyl benzoate, diethyl succinate, ethyl decanoate and ethyl laurate. In total, 11 and three released bound volatiles were found in orange juice and wine, respectively, and most of them were not found in free form. Only ethyl 3-hydroxyhexanoate and cis -carveol were found present in both the free and bound forms of orange juice. Copyright 2009 John Wiley & Sons, Ltd. [source]

Analysis of carbohydrates and amino acids in vegetable waste waters by ion chromatography

Michele Arienzo
Abstract High-performance anion exchange chromatography coupled with pulsed amperometric detection was used for the quantitative determination of total and free sugars in olive oil mill waste waters (OMWW). Automated amino acid ion chromatography was employed to analyse total and free amino acids in the same OMWW. Sugars were analysed in samples pre-purified by means of a three-step purification procedure involving: (i) methanol precipitation of OMWW; (ii) dialysis of the obtained solid and liquid fractions; and (iii) chromatographic purification on RP18 phase followed by Amberlite resin. The amino acids were determined directly in samples obtained from the first two steps performed for sugar analysis. The analysis carried out with the reported methodologies allowed the quantitative determination of total sugars and amino acids and the differentiation between their free and bound forms. The sugars determined were arabinose, fructose, galactose, glucose, rhamnose, xylose, galacturonic and glucuronic acids, and the amino acids were Asp, Glu, Thr, Ser, Pro, Gly, Ala, Val, Met, Ile, Leu, Tyr, Phe, Lys, His, Arg and Cys. Asn, Gln, and Trp were not detected. The technological, biotechnological and environmental advantages arising from this analytical methodology applied to OMWW are briefly discussed. Copyright 2003 John Wiley & Sons, Ltd. [source]

Structures of S. aureus thymidylate kinase reveal an atypical active site configuration and an intermediate conformational state upon substrate binding

Masayo Kotaka
Abstract Methicillin-resistant Staphylococcus aureus (MRSA) poses a major threat to human health, particularly through hospital acquired infection. The spread of MRSA means that novel targets are required to develop potential inhibitors to combat infections caused by such drug-resistant bacteria. Thymidylate kinase (TMK) is attractive as an antibacterial target as it is essential for providing components for DNA synthesis. Here, we report crystal structures of unliganded and thymidylate-bound forms of S. aureus thymidylate kinase (SaTMK). His-tagged and untagged SaTMK crystallize with differing lattice packing and show variations in conformational states for unliganded and thymidylate (TMP) bound forms. In addition to open and closed forms of SaTMK, an intermediate conformation in TMP binding is observed, in which the site is partially closed. Analysis of these structures indicates a sequence of events upon TMP binding, with helix ,3 shifting position initially, followed by movement of ,2 to close the substrate site. In addition, we observe significant conformational differences in the TMP-binding site in SaTMK as compared to available TMK structures from other bacterial species, Escherichia coli and Mycobacterium tuberculosis as well as human TMK. In SaTMK, Arg 48 is situated at the base of the TMP-binding site, close to the thymine ring, whereas a cis -proline occupies the equivalent position in other TMKs. The observed TMK structural differences mean that design of compounds highly specific for the S. aureus enzyme looks possible; such inhibitors could minimize the transfer of drug resistance between different bacterial species. [source]

Structure of Escherichia coli YfdW, a type III CoA transferase

Arhonda Gogos
Crystal structures are reported for free and coenzyme A (CoA) bound forms of the YfdW protein from Escherichia coli, a representative type III CoA transferase. The structures reveal a two-domain protomer with interdomain connections forming a ring-like structure with a large central hole. Two protomers associate to form a highly intertwined dimer in which the hole of each ring is filled by the partner molecule. Each protomer binds a single CoA molecule and these CoA-binding sites are distant from one another in the dimer. [source]

Cloning, expression and crystallization of dihydrodipicolinate reductase from methicillin-resistant Staphylococcus aureus

Sudhir Dommaraju
Dihydrodipicolinate reductase (DHDPR; EC catalyzes the nucleotide (NADH/NADPH) dependent second step of the lysine-biosynthesis pathway in bacteria and plants. Here, the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DHDPR from methicillin-resistant Staphylococcus aureus (MRSA-DHDPR) are presented. The enzyme was crystallized in a number of forms, predominantly with ammonium sulfate as a precipitant, with the best crystal form diffracting to beyond 3.65, resolution. Crystal structures of the apo form as well as of cofactor (NADPH) bound and inhibitor (2,6-pyridinedicarboxylate) bound forms of MRSA-DHDPR will provide insight into the structure and function of this essential enzyme and valid drug target. [source]