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BBA - Proteins and Proteomics (v.1774, #8)
Enhanced preference for π-bond containing substrates is correlated to Pro110 in the substrate-binding tunnel of Escherichia coli thioesterase I/protease I/lysophospholipase L1 by Li-Chiun Lee; Yen-Chywan Liaw; Ya-Lin Lee; Jei-Fu Shaw (pp. 959-967).
Escherichia coli thioesterase I/protease I/lysophospholipase L1 (TAP) possesses multifunctional enzyme with thioesterase, esterase, arylesterase, protease, and lysophospholipase activities. Leu109, located at the substrate-binding tunnel, when substituted with proline (Pro) in TAP, shifted the substrate-preference from medium-to-long acyl chains to shorter acyl chains of triglyceride and p-nitrophenyl ester, and increased the preference for aromatic-amino acid-derived esters. In the three-dimensional TAP structures, the only noticeable alteration of backbone and side chain conformation was located at the downstream Pro110–Ala123 region rather than at Pro109 itself. The residue Pro110, adjacent to Leu109 or Pro109, was found to contribute to the substrate preference of TAP enzymes for esters containing acyl groups with π bond(s) or aromatic group(s). Some of the interactions between the enzyme protein and the substrate may be contributed by an attractive force between the Pro110 C–H donor and the substrate π-acceptor.
Keywords: Proline substituent; Substrate-binding tunnel; Substrate specificity; Enzyme kinetics; π bond
A proteomic study of sodium/d-glucose cotransporter 1 (SGLT1): Topology of loop 13 and coverage of other functionally important domains by Azad Kumar; Navneet K. Tyagi; Enrique Arevalo; Keith W. Miller; Rolf K.H. Kinne (pp. 968-974).
In order to obtain further information about the structure and function of human sodium/d-glucose cotransporter 1 (hSGLT1), the recombinant protein was subjected, either after reconstitution into liposomes or in its free form, to proteolysis followed by nanoscale microcapillary liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). The peptides released from SGLT1 proteoliposomes by trypsin bead digestion represented the early N-terminal, loop 7, and loop 9, supporting topology models that place these domains on the extracellular side of the protein. Trypsin bead digestion generated, however, also a number of peptides derived from loop 13 whose topology with regard to the membrane is hitherto a point of debate. Sequence coverage was provided from amino acids 559 to 644, suggesting that loop 13 is almost completely accessible at the extravesicular face of the proteoliposomes. These results support the notion that major parts of loop 13, essential for the interaction with transport inhibitors in vivo, are located extracellularly in intact cells. In-gel trypsin, chymotrypsin, and in particular trypsin/chymotrypsin digestion of recombinant SGLT1 in combination with LC-MS/MS provide extensive sequence coverage of the protein, including domains involved in sugar and inhibitor binding and potential phosphorylation sites. These studies demonstrate that proteomic analysis combined with mass spectrometry is a useful tool to characterize regions of SGLT1 that are important for its function and regulation.
Keywords: Abbreviations; SGLT1; sodium/; d; -glucose cotransporter 1; LC-MS/MS; nanoscale microcapillary liquid chromatography electrospray ionization tandem mass spectrometry; ACN; acetonitril; TFA; trifluoroacetic acid; aas; amino acid sequence; m; /; z; mass to charge ratioSodium/; d; -glucose cotransporter 1; LC-MS/MS; Membrane topology; d; -glucose; Phlorizin
Quantitative fluorometric analysis of the protective effect of chitosan on thermal unfolding of catalytically active native and genetically-engineered chitosanases by Sébastien Roy; Mélanie Fortin; Julie Gagnon; Mariana Gabriela Ghinet; Jean-Guy LeHoux; Gilles Dupuis; Ryszard Brzezinski (pp. 975-984).
We have taken advantage of the intrinsic fluorescence properties of chitosanases to rapidly and quantitatively evaluate the protective effect of chitosan against thermal denaturation of chitosanases. The studies were done using wild type chitosanases N174 produced by Streptomyces sp. N174 and SCO produced by Streptomyces coelicolor A3(2). In addition, two mutants of N174 genetically engineered by single amino acid substitutions (A104L and K164R) and one “consensus” (N174-CONS) chitosanase designed by multiple amino acid substitutions of N174 were analyzed. Chitosan used had a weight average molecular weight ( Mw) of 220 kDa and was 85% deacetylated. Results showed a pH and concentration-dependent protective effect of chitosan in all the cases. However, the extent of thermal protection varied depending on chitosanases, suggesting that key amino acid residues contributed to resistance to heat denaturation. The transition temperatures ( Tm) of N174 were 54 °C and 69.5 °C in the absence and presence (6 g/l) of chitosan, respectively. Tm were increased by 11.6 °C (N174-CONS), 13.8 °C (CSN-A104L), 15.6 °C (N174-K164R) and 25.2 °C (SCO) in the presence of chitosan (6 g/l). The thermal protective effect was attributed to an enzyme–ligand thermostabilization mechanism since it was not mimicked by the presence of anionic (carboxymethyl cellulose, heparin) or cationic (polyethylene imine) polymers, polyhydroxylated (glycerol, sorbitol) compounds or inorganic salts. Furthermore, the data from fluorometry experiments were in agreement with those obtained by analysis of reaction time-courses performed at 61 °C in which case CSN-A104L was rapidly inactivated whereas N174, N174-CONS and N174-K164R remained active over a reaction time of 90 min. This study presents evidence that (1) the fluorometric determination of Tm in the presence of chitosan is a reliable technique for a rapid assessment of the thermal behavior of chitosanases, (2) it is applicable to structure–function studies of mutant chitosanases and, (3) it can be useful to provide an insight into the mechanism by which mutations can influence chitosanase stability.
Keywords: Abbreviations; GH; glycoside hydrolases; Mw; weight average molecular weight; N174; chitosanase from; Streptomyces; sp. N174; N174-CONS; consensus chitosanase sequence derived from N174; PEI; polyethyleneimine; SCO; chitosanase SCO0677 from; Streptomyces coelicolor; A3(2); T; m; transition temperature; U; unitChitosanase; Mutagenesis; Thermostability; Fluorometry; Chitosan
Thermodynamic and kinetic characterization of the association of triosephosphate isomerase: The role of diffusion by Hugo Nájera; Leonardo Dagdug; D. Alejandro Fernández-Velasco (pp. 985-994).
It is known that diffusion plays a central role in the folding of small monomeric proteins and in the rigid-body association of proteins, however, the role of diffusion in the association of the folding intermediates of oligomeric proteins has been scarcely explored. In this work, catalytic activity and fluorescence measurements were used to study the effect of viscosity in the unfolding and refolding of the homodimeric enzyme triosephosphate isomerase from Saccharamyces cerevisiae. Two transitions were found by equilibrium and kinetic experiments, suggesting a three-state model with a monomeric intermediate. Glycerol barely affects Δ G0fold whereas Δ G0assoc becomes more favourable in the presence of the cosolvent. From 0 to 60% (v/v) glycerol, the association rate constant showed a near unitary dependence on solvent viscosity. However, at higher glycerol concentrations deviations from Kramers theory were observed. The dissociation rate constant showed a viscosity effect much higher than one. This may be related to secondary effects such as short-range glycerol-induced repulsion between monomers. Nevertheless, after comparison under isostability conditions, a slope near one was also observed for the dissociation rate. These results strongly suggest that the bimolecular association producing the native dimer is limited by diffusional events of the polypeptide chains through the solvent.
Keywords: Abbreviations; TIM; Triosephosphate isomerase; yTIM; TIM from; Saccharomyces cerevisiae; GdmCl; Guanidinium chloride; SCM; Spectral Centre of MassDimer; Diffusion; Glycerol; Protein stability; TIM barrel
Preceding hydrophobic and β-branched amino acids attenuate splicing by the CnePRP8 intein by Esther J. Pearl; Annika A.M. Bokor; Margi I. Butler; Russell T.M. Poulter; Sigurd M. Wilbanks (pp. 995-1001).
As the Cne PRP8 intein is active and exists in an essential gene of an important fungal pathogen, inhibitors of splicing and assays for intein activity are of interest. The self-splicing activity of Cne PRP8, the intein from the Prp8 gene of Cryptococcus neoformans, was assessed in different heterologous fusion proteins expressed in Escherichia coli. Placement of a putatively inactive variant of the intein adjacent to the α-complementation peptide abolished the peptide's ability to restore β-galactosidase activity, while an active variant allowed complementation. This α-complementation peptide therefore provides a facile assay of splicing which can be used to test potential inhibitors. When placed between two heterologous protein domains, splicing was impaired by a β-branched amino acid immediately preceding the intein, while splicing occurred only with a hydroxyl or thiol immediately following the intein. Both these assays sensitively report impairment of splicing and provide information on how context constrains the splicing ability of Cne PRP8.
Keywords: Abbreviations; CBD; chitin binding domain; Cne PRP8; the intein of the; C. neoformans Prp8; gene; HiTF; H. influenzae; trigger factor protein; IPTG; isopropyl β-; d; -thiogalactopyranoside; Psp Pol1; the intein of; Pyrococcus sp.; GB-D polymerase; Psp DnaE; the intein of; Synechococcus; sp 6803 DNA polymerase; VMA; the vacuolar membrane ATPaseProtein splicing; Intein; Cryptococcus
Molecular dynamics simulation study of interaction between a class IIa bacteriocin and its immunity protein by Wael Soliman; Subir Bhattacharjee; Kamaljit Kaur (pp. 1002-1013).
Molecular dynamics (MD) simulations of carnobacteriocin B2 (CbnB2), a structurally well-characterized class IIa bacteriocin, and its immunity protein (ImB2) in lipid bilayer environment have been conducted to explore the interaction between them. Six 30-ns simulations were conducted in DPPC or POPG bilayer systems. In these simulations, ImB2 was placed in the aqueous layer with different orientations facing CbnB2 to sample all the faces of ImB2. The MD results indicate that (i) while CbnB2 remained embedded in the bilayer, it tends to move toward the interface, and (ii) the presence of CbnB2 in the DPPC bilayer attracts ImB2 toward the bilayer. In one of the orientations in DPPC bilayer system (simulation 1), ImB2 penetrates the bilayer and interacts with CbnB2 by ion-pair interaction. At several instances toward the later half of the simulation (15–30 ns), ImB2 and CbnB2 were found to form salt-bridge between Arg95 of ImB2 and Glu24 of CbnB2. Simulation in POPG bilayer displayed strong interaction between the positively charged ImB2 and the negatively charged polar head groups of the POPG molecules at the lipid–water interface. However, ImB2 was not able to penetrate the bilayer thereby preventing any interaction between ImB2 and CbnB2.
Keywords: Abbreviations; Asp; aspartic acid; CbnB2; carnobacteriocin B2; DPPC; dipalmitoylphosphatidylcholine; e; electronic charge (1.602; ×; 10; −; 19; C); His; histidine; ImB2; carnobacteriocin B2 immunity protein; LeuA; leucocin A; Lys; lysine; MD; molecular dynamics; NMR; nuclear magnetic resonance; ns; nanosecond; pbc; periodic boundary conditions; PDB; protein data bank; PME; particle mesh Ewald; POPG; palmitoyloleoylphosphatidylglycerol; Pro; proline; ps; picosecond; RMSD; root mean square deviation; SakP; sakacin P; SPC; simple point chargeClass IIa bacteriocin; Carnobacteriocin B2; Immunity protein; Peptide–peptide interaction; Lipid bilayer; MD simulation
6His–Eco29kI methyltransferase methylation site and kinetic mechanism characterization by Dmitri Nikitin; Marina Mokrishcheva; Alexander Solonin (pp. 1014-1019).
A new type II 6His–Eco29kI DNA methyltransferase was tested for methylation site (CCMeGCGG) and catalytic reaction optimal conditions. With high substrate concentrations, an inhibitory effect of DNA, but not AdoMet, on its activity was observed. Isotope partitioning and substrate preincubation assays showed that the enzyme–AdoMet complex is catalytically active. Considering effect of different concentrations of DNA and AdoMet on initial velocity, ping-pong mechanisms were ruled out. According to data obtained, the enzyme appears to work by preferred ordered bi–bi mechanism with AdoMet as leading substrate.
Keywords: Abbreviations; AdoMet; S-adenosyl-; l; -methionine; RMS; restriction–modification system; MTase; DNA-methyltransferase; RE; restriction endonuclease; 6His; 6 Histidine residues; LB medium; Luria–Bertani medium; Rpm; rotations per minute; DE; diethylaminoethyl; Ni-NTA; Nickel nitrilo-tri-acetic acid; EDTA; Ethyl diaminotetraacetate; DTT; dithiotreitol; PAGE; polyacrylamide gel electrophoresis; Vol; volume; U; enzyme activity units; μg; 10; −6; g; hr (hrs); hour (hours); sec; secondDNA methyltransferase; Methylation site detection; Kinetic mechanism; Substrate inhibition
Venom phospholipases of Russell's vipers from Myanmar and eastern India—Cloning, characterization and phylogeographic analysis by Inn-Ho Tsai; Hsin-Yu Tsai; Ying-Ming Wang; Tun-Pe; David A. Warrell (pp. 1020-1028).
Venoms of Russell's vipers (genus Daboia) are known for their deadly coagulopathic and other effects. We herein studied various isoforms of venom phospholipases A2 (PLAs) from two Daboia species at their geographic boundary. From Myanmar Daboia siamensis venom (designated as DsM), four PLAs (designated DsM-aI, aI', aII' and bI') were purified, and the cDNAs encoding two acidic (DsM-aI and aII) and two basic PLAs (DsM-bI and S1) were also cloned from its venom-glands. DsM-S1 is identical to the major venom PLA of southern India Daboia russelii, but the protein is absent from the venom. Additionally, four PLAs (designated DrK-aI, aII, bI and bII) were cloned from cDNA obtained from venom glands of a Kolkata D. russelii, and the PLAs were purified from the pooled venom (designated as DrK). The acidic DrK-aI is the most neurotoxic and lethal among these PLAs; DsM-aI which differs from DrK-aI by only the Phe2 substitution shows greatly reduced enzymatic activity and lethality. Both acidic PLAs do not form dimeric complex with basic PLAs in the same venoms. DsM-bI' is neurotoxic and lethal but its orthologous DrK-bI (97% identical to DsM-bI') is a much weaker toxin. Given the fact that most of the orthologous PLAs of DrK and DsM share 97–100% sequence identity, Daboia vipers of Myanmar and Kolkata must be closely related. Molecular phylogenetic analyses on 30 venom PLAs of Eurasian vipers' revealed co-evolution of five subtypes of venom PLAs in both Daboia and Vipera genera. Our results shed light on the intra- and inter-species variations and structure–function relationships of viperid venom PLAs.
Keywords: Abbreviations; DsM; Daboia siamensis; (Myanmar); DrK; Daboia russelii; (Kolkata); dPPC; l; -dipalmitoyl phosphatidylcholine; PLA; Phospholipase A; 2; HPLC; high performance liquid chromatographyVenom phospholipase A; 2; Cloning and sequencing; Phylogenetic analysis; Geographic variation; Russell's viper (; Daboia siamensis, Daboia russelii; )
Role of amino acid residue 90 in bioactivity and receptor binding capacity of tumor necrosis factor mutants by Hiroko Shibata; Haruhiko Kamada; Kyoko Kobayashi-Nishibata; Yasuo Yoshioka; Toshihide Nishibata; Yasuhiro Abe; Tetsuya Nomura; Hiromi Nabeshi; Kyoko Minowa; Yohei Mukai; Shinsaku Nakagawa; Tadanori Mayumi; Shin-ichi Tsunoda; Yasuo Tsutsumi (pp. 1029-1035).
We have previously produced two bioactive lysine-deficient mutants of TNF-α (mutTNF-K90R,-K90P) and found that these mutants have bioactivity superior to wild-type TNF (wtTNF). Because these mutants contained same amino acid except for amino acid 90, it is unclear which amino acid residue is optimal for showing bioactivity. We speculated that this amino acid position was exchangeable, and this amino acid substitution enabled the creation of lysine-deficient mutants with enhanced bioactivity. Therefore, we produced mutTNF-K90R variants (mutTNF-R90X), in which R90 was replaced with other amino acids, to assay their bioactivities and investigated the importance of amino acid position 90. As a result, mutTNF-R90X that replaced R90 with lysine, arginine and proline were bioactive, while other mutants were not bioactive. Moreover, these three mutants showed bioactivity as good as or better than wtTNF. R90 replaced with lysine or arginine had especially superior binding affinities. These results suggest that the amino acid position 90 in TNF-α is important for TNF-α bioactivity and could be altered to improve its bioactivity to generate a “super-agonist”.
Keywords: TNF; Mutant; Phage display; Lysine residue; TNF receptors; Structure
The neck of bacteriophage T4 is a ring-like structure formed by a hetero-oligomer of gp13 and gp14 by Tahmina Akhter; Li Zhao; Atsushi Kohda; Kazuhiro Mio; Shuji Kanamaru; Fumio Arisaka (pp. 1036-1043).
After packaging of DNA into the head of bacteriophage T4 is completed, a neck is formed at the portal vertex of the head to be ready for the tail attachment. The main components of the neck are gp13 and gp14 (gp: gene product), which consist of 309 and 256 amino acid residues, respectively. In order to elucidate the structure and subunit arrangement in the neck, overexpression systems of gene 13 and gene 14 were constructed and purified to homogeneity. Far-UV circular dichroism (CD) spectra of gp13 and gp14 indicated that gp13 is rich in α-helices whereas gp14 is rich in β-sheets. Sedimentation velocity analysis of gp13 and gp14 revealed that both proteins are present as monomers in solution. The frictional ratios ( f/ f0) of the two proteins indicated that gp14 has a more elongated shape than gp13. Although isolated gp13 and gp14 do not interact with each other when mixed under physiological conditions, they form a hetero-oligomer complex with the stoichiometry of 10:5 after treatment with ammonium sulfate. Electron microscopy of this complex has shown that it forms a ring-like structure of 15 nm in diameter.
Keywords: Abbreviations; bp; base pair; CBB; Coomassie Brilliant Blue; EDTA; ethylenediaminetetraacetic acid; TEM; transmission electron microscope; gp; gene product; IPTG; isopropyl β-; d; -1-thiogalactopyranoside; PCR; polymerase chain reaction; PMSF; phenylmethylsulphonyl fluoride; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis; PVDF; polyvinylidine difluorideBacteriophage T4; Neck protein; Virus assembly; Protein–protein interaction; Analytical ultracentrifugation; Transmission electron microscopy
Hippocampal signaling protein levels are different in early and late metestrus in the rat by Wei-Fei Diao; Harald Höger; Wei-Qiang Chen; Arnold Pollak; Gert Lubec (pp. 1044-1051).
Early and late metestrus in the rat differ by progesterone levels. As it is known that progesterone shows a potential negative effect on cognitive performances and can counteract the estradiol-induced neural effects, we intended to study signaling proteins in the hippocampus, a structure representing a main brain area of cognitive function. Female OFA Sprague–Dawley rats were used in the studies and estrous phases were determined using vaginal smears. Hippocampal tissue was taken, proteins extracted, run on two-dimensional gel electrophoresis and proteins were identified by mass spectrometry methods (MALDI-TOF-TOF and nano-LC-ESI-MS/MS). Individual signaling protein levels quantified by specific software were shown to vary between the two phases, including NG,NG-dimethylarginine dimethylaminohydrolase 1 for nitric oxide signaling, guanine nucleotide-binding proteins, septin 6, septin 11, G-septin alpha, and 14-3-3 protein gamma. Results from this study indicate that early and late metestrus show differences in signaling pathways, that may help to design further investigations at the protein level and may assist to interpret literature on protein expression and brain protein levels in female rats. Moreover, signaling differences in hippocampus are challenging cognitive studies during these two metestrus phases probably revealing cognitive differences between early and late metestrus.
Keywords: Estrous; Mass spectrometry; Protein expression; Rat; Sex hormone; Two-dimensional gel electrophoresis
Solvent as a competitive inhibitor for Candida antarctica lipase B by Marianne Graber; Romain Irague; Eric Rosenfeld; Sylvain Lamare; Linda Franson; Karl Hult (pp. 1052-1057).
In enzyme-catalyzed reactions, the choice of solvent often has a marked effect on the reaction outcome. In this paper, it is shown that solvent effects could be explained by the ability of the solvent to act as a competitive inhibitor to the substrate. Experimentally, the effect of six solvents, 2-pentanone, 3-pentanone, 2-methyl-2-pentanol, 3-methyl-3-pentanol, 2-methylpentane and 3-methylpentane, was studied in a solid/gas reactor. As a model reaction, the CALB-catalyzed transacylation between methyl propanoate and 1-propanol, was studied. It was shown that both ketones inhibited the enzyme activity whereas the tertiary alcohols and the hydrocarbons did not. Alcohol inhibition constants, KiI were changed to "Ki", determined in presence of 2-pentanone, 3-pentanone, and 3-methyl-3-pentanol, confirmed the marked inhibitory character of the ketones and an absence of inhibition of 3-methyl-3-pentanol. The molecular modeling study was performed on three solvents, 2-pentanone, 2-methyl-2-pentanol and 2-methyl pentane. It showed a clear inhibitory effect for the ketone and the tertiary alcohol, but no effect for the hydrocarbon. No change in enzyme conformation was seen during the simulations. The study led to the conclusion that the effect of added organic component on lipase catalyzed transacylation could be explained by the competitive inhibitory character of solvents towards the first binding substrate methyl propanoate.
Keywords: Kinetics; Organic solvent; Molecular modeling; Solid/gas biocatalysis; Conformational change; Solubility
Molecular docking and spatial coarse graining simulations as tools to investigate substrate recognition, enhancer binding and conformational transitions in indoleamine-2,3-dioxygenase (IDO) by Antonio Macchiarulo; Roberto Nuti; Daniele Bellocchi; Emidio Camaioni; Roberto Pellicciari (pp. 1058-1068).
Indoleamine 2,3-dioxygenase (IDO) is an heme-containing enzyme involved in the regulation of important immunological responses and neurological processes. The enzyme catalyzes the oxidative cleavage of the pyrrole ring of the indole nucleus of tryptophan (Trp) to yield N-formylkynurenine, that is the initial and rate limiting step of the kynurenine pathway. Some indole derivatives have been reported to act as effectors of the enzyme by enhancing its catalytic activity. On the basis of the recent availability of the crystal structure of IDO, in this work we investigate substrate recognition and enhancer binding to IDO using molecular docking experiments. In addition, conformational transitions of IDO in response to substrate and enhancer binding are studied using coarse graining simulations with the program FIRST. The results enable us to identify (i) the binding site of enhancer modulators; (ii) the motion of an electrostatic gate that regulates the access of the substrate to the catalytic site of the enzyme; (iii) the movement of the anchoring region of a hairpin loop that may assist the shuttle of substrates/products to/from the catalytic site of IDO. These data, combined with available site-directed mutagenesis experiments, reveal that conformational transitions of IDO in response to substrate and enhancer binding are controlled by distinct combination of two conformational states (open and close) of the above structural motifs. On this basis, a molecular mechanism regarding substrate recognition and activity enhancement by indole derivatives is proposed.
Keywords: IDO; Docking; Spatial coarse graining simulation; Enhancer; Substrate recognition