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BBA - Proteins and Proteomics (v.1752, #2)
Cooperativity in the motor activities of the ATP-fueled molecular motors by Ming S. Liu; B.D. Todd; Richard J. Sadus (pp. 111-123).
Kinesin, myosin and F1-ATPase are multi-domain molecular motors with multiple catalytic subunits. The motor mechanochemics are achieved via the conversion of ATP hydrolysis energy into forces and motions. We find that the catalysis of these molecular motors do not follow the simple Michaelis–Menten mechanism. The motor activities, such as the hydrolysis or processive rates, of kinesin, myosin and F1-ATPase have a complex ATP-dependent cooperativity. To understand this complexity in kinetics and mechanochemics, we develop a conformation correlation theory of cooperativity for the ATP-fueled motor proteins. The quantitative analysis and simulations indicate that cooperativity is induced by the conformational coupling of binding states of different subunits and prevails in the motor activities.
Keywords: Molecular motor; Cooperativity; Motor activity; ATPase catalysis; Conformation correlation; Binding change
Analysis of protein–surfactant interactions—a titration calorimetric and fluorescence spectroscopic investigation of interactions between Humicola insolens cutinase and an anionic surfactant by Anders D. Nielsen; Lise Arleth; Peter Westh (pp. 124-132).
We have studied interactions of cutinase (HiC) from Humicula insolens and sodium dodecyl sulphate (SDS) by parallel calorimetric and fluorescence investigations of systems in which the concentration of both components was changed systematically. Results from the two methods exhibit a number of synchronous characteristics, when plotted against the total SDS concentration, [SDS]tot. The molecular origin of several of these anomalies was assigned, and five intervals of [SDS]tot in which different modes of interactions dominated were identified. Going from low to high [SDS]tot, these modes were: binding of (a few) SDS to native HiC, formation of oligomeric protein aggregates, denaturation of HiC and adsorption of SDS on denatured protein. For [SDS]tot>3–6 mM (depending on the protein concentration), the adsorption saturated, and no further protein–detergent interaction could be detected.Two particularly conspicuous anomalies in the calorimetric data were ascribed to respectively denaturation and saturation. It was found that [SDS]tot at these points depended linearly on the (total) protein concentration, [HiC]. We suggest that this reflects the balance between bound and free SDS [SDS]tot=[SDS]aq+[HiC] Nb where [SDS]aq and Nb are, respectively, the aqueous (“free�) concentration of SDS and the average number of SDS bound per protein. Interpretation of the results along these lines showed that at 22 °C and pH 7.0, HiC denatures with ∼14 bound surfactant molecules at [SDS]aq=1.0 mM. Saturation is characterized by Nb ∼39 and [SDS]aq=2.2 mM. The latter value is equal to CMC in the (protein free) buffer. These results are discussed with respect to the SDS-binding capacity of HiC and the origin and location of the saturation point.
Keywords: Abbreviations; HiC; Humicola insolens; cutinase; FsC; Fusarium solani pisi; cutinase; ITC; isothermal titration calorimetry; MR; molar ratio; CMC; critical micelle concentration; SDS; sodium dodecyl sulfate; 2,6-TNS; 6-(p-Toluidino)-2-naphthalenesulfonic acid; 1,8-ANS; 8-Anilino-1-naphthalenesulfonic acid; AOT; Bis(2-etylhexyl) sodium sulfosuccinate; BSA; bovine serum albuminSDS; Thermodynamics; Denaturation; Saturation; Apparent CMC
Cloning and characterization of an Arabidopsis thaliana Nudix hydrolase homologous to the mammalian GFG protein by Kamil Olejnik; Elzbieta Kraszewska (pp. 133-141).
In a search for a plant antimutator MutT protein, an Arabidopsis thaliana Nudix hydrolase with homology to the mammalian GFG protein was expressed as a hexahistidine fusion polypeptide in Escherichia coli and purified to homogeneity. Unlike the GFG protein, the A. thaliana homolog could not complement the mutT mutation in a MutT-deficient E. coli strain nor was it able to hydrolyze 8-oxo-dGTP, the main substrate of the MutT protein. Instead the recombinant protein hydrolyzed a variety of nucleoside diphosphate derivatives showing a preference for ADP-ribose, with Km and kcat values of 1.2 mM and 2.7 s−1 respectively. The products of ADP-ribose hydrolysis were AMP and ribose-5-phosphate. The optimal activity was at alkaline pH (8.5) with Mg2+ (5 mM) ions as the cofactor. The protein exists as a dimmer in solution.
Keywords: Plant; Arabidopsis thaliana; Antisense fibroblast growth factor; GFG protein; Nudix hydrolase; ADP-ribose
Thermostability and in vitro digestibility of a purified major allergen 2S albumin (Ses i 1) from white sesame seeds ( Sesamum indicum L.) by F. Javier Moreno; Bárbara M. Maldonado; Nikolaus Wellner; E.N. Clare Mills (pp. 142-153).
A major 2S albumin allergen, Ses i 1, from white sesame seeds was purified to homogeneity, characterized and identified using proteomic techniques. Ses i 1 exhibited a molecular weight of 12062 Da, although an extensive C-terminal clipping of the small subunit was observed. In addition, the N-terminal glutamine of the small subunit had been converted to pyroglutamate and a variant of the large subunit which had lost the N-terminal glutamine was also detected. The protein was thermo-stable up to 90 °C at neutral and acid pH, retaining its monomeric state and showing minimal alterations, which were reversible on cooling, in a predominantly α-helical secondary structure, as shown by circular dichroism and Fourier transform-infrared spectroscopy. Ses i 1 was also highly resistant to digestion using a physiologically relevant in vitro gastrointestinal model system. After 2 h of gastric digestion, the allergen remained completely intact and only the small subunit was cleaved during 2 h of subsequent duodenal digestion, leaving a major IgE epitope region of this protein intact. Neither prior heating of the Ses i 1 nor the presence of the physiological surfactant phosphatidylcholine affected the pattern of proteolysis. These findings are consistent with those found for the 2S albumin allergen from Brazil nut, Ber e 1, and suggest that Ses i 1 may preserve its structure from the degradation in the gastrointestinal tract, a property thought to be crucial for both a protein to sensitise the mucosal immune system and provoke an allergic reaction in a sensitised individual.
Keywords: Food allergy; 2S albumin; Sesame; Digestion; Proteomic
Complete refolding of bovine β-lactoglobulin requires disulfide bond formation under strict conditions by Makoto Hattori; Kazuhiko Hiramatsu; Takashi Kurata; Mika Nishiura; Koji Takahashi; Akio Ametani; Shuichi Kaminogawa (pp. 154-165).
β-Lactoglobulin (β-LG) denatured with 6 M guanidine hydrochloride (GdnHCl) containing a reducing agent and subsequently dialysed against phosphate-buffered saline (PBS) resulted in incomplete refolding of this protein despite the fact that the biological activity for retinol-binding was recovered to almost the same degree as that of the native molecule [Hattori, M., Ametani, A., Katakura, Y., Shimizu, M., Kaminogawa, S. J., Biol. Chem. 268 (1993) 22414–22419]. The enzyme probe method, evaluation of hydrophilicity values, in-gel mobility on SDS-PAGE, and evaluation of disulfide bonds with the Ellman method showed exposure of the hydrophobic region(s) and incorrect disulfide bond formation in such dialyzed β-LG molecules. We reveal in this present work that complete refolding could be attained by diluting denatured β-LG with PBS containing a reducing agent, before slow reoxidation of the sulfhydryl groups upon dialysis for gradient removal of the reducing agent in 6 steps. Complete renaturation was confirmed by analyzing the retinol-binding activity, CD spectra, intrinsic fluorescence, binding ability of monoclonal antibodies (mAbs), and SDS-PAGE. Step-by-step disulfide bond formation was considered to be critical for the complete refolding of denatured β-LG. Our method can contribute to establish a procedure for complete refolding of useful recombinant proteins in vitro without such biological aids as chaperones.
Keywords: Abbreviations; β-LG; β-lactoglobulin; RCM-β-LG; reduced and carboxymethylated β-lactoglobulin; PBS; phosphate-buffered saline; GSH; reduced form of glutathione; GSSG; oxidized form of glutathione; K; AS; equilibrium constant; K′; d; dissociation constant; mAb; monoclonal antibody; MES; 2-(; N; -morpholino) ethanesulfonic acid; GdnHCl; guanidine hydrochlorideβ-Lactoglobulin; Disulfide bond formation; Refolding
Differential expression of the fast skeletal muscle proteome following chronic low-frequency stimulation by Pamela Donoghue; Philip Doran; Paul Dowling; Kay Ohlendieck (pp. 166-176).
Physiological and biochemical responses of skeletal muscle fibres to enhanced neuromuscular activity under conditions of maximum activation can be studied experimentally by chronic low-frequency stimulation of fast muscles. Stimulation-induced changes in the expression pattern of the rabbit fast skeletal muscle proteome were evaluated by two-dimensional gel electrophoresis and compared to the altered isoform expression profile of established transformation markers such as the Ca2+-ATPase, calsequestrin and the myosin heavy chain. Sixteen muscle proteins exhibited a marked change in their expression level. This included albumin with a 4-fold increase in abundance. In contrast, glycolytic enzymes, such as enolase and aldolase, showed a decreased expression. Concomitant changes were observed with marker elements of the contractile apparatus. While the fast isoforms of troponin T and myosin light chain 2 were drastically down-regulated, their slow counterparts exhibited increased expression. Interestingly, mitochondrial creatine kinase expression increased while the cytosolic isoform of this key muscle enzyme decreased. The expression of the small heat shock protein HSP-B5/αB-crystallin and the oxygen carrier protein myoglobin were both increased 2-fold following stimulation. The observed changes indicate that the conversion into fatigue-resistant red fibres depends on: (i) the optimum utilization of free fatty acids via albumin transportation, (ii) a rearrangement of the creatine kinase isozyme pattern for enhanced mitochondrial activity, (iii) an increased availability of oxygen for aerobic metabolism via myoglobin transport, (iv) the conversion of the contractile apparatus to isoforms with slower twitch characteristics and (v) the up-regulation of chaperone-like proteins for stabilising myofibrillar components during the fast-to-slow transition process.
Keywords: Skeletal muscle proteome; Chronic low-frequency stimulation; Muscle transformation; Muscle plasticity; Muscle proteomics
Crystal structure of the complex of the secretory phospholipase A2 from Daboia russelli pulchella with an endogenic indole derivative, 2-carbamoylmethyl-5-propyl-octahydro-indol-7-yl-acetic acid at 1.8 Å resolution by R. Balasubramanya; Vikas Chandra; Punit Kaur; Tej P. Singh (pp. 177-185).
Phospholipase A2 (PLA2) enzymes from snake venoms are approximately 14 kDa secretory proteins and catalyze the release of arachidonic acid which is the precursor of proinflammatory mediators such as prostaglandins, leukotrienes, thromboxanes and platelet-activating factors. The structure of the PLA2 enzyme purified from the venom of Daboia russelli pulchella was determined using molecular replacement method and refined to an R value of 18.3% for all the reflections to 1.8 Å resolution. The structure contains two crystallographically independent molecules A and B which form an asymmetric homodimer. The Ca2+ ion was not detected in the present structure, however, a characteristic non-protein high quality electron density was observed at the substrate-binding site of molecule A which allowed a clear interpretation of a natural ligand identified as a derivative of indole, 2-carbamoylmethyl-5-propyl-octahydro-indol-7-yl)-acetic acid. The corresponding substrate-binding site in molecule B was empty. The ligand present in molecule A is involved in extensive interactions with the protein atoms including important catalytic residues such as Asp-49 and His-48. The results also show that the indole derivatives act as potent inhibitors of secretory group II PLA2 enzymes that can be further modified to be used as potential therapeutic agents.
Keywords: Abbreviations; indole; 2-carbamoylmethyl-5-propyl-octahydro-indol-7-yl)-acetic acid; Phospholipase A; 2; PLA; 2; DRP; Daboia russelli pulchella; DLS; dynamic light scattering; MALDI-TOF; matrix assisted laser desorption ionization-time of flight; CCP4i; Collaborative Computational Project Number 4, interactive; R; H; hydrodynamic radius, r.m.s. root mean square; hnps; human non pancreatic secretoryPhospholipase A; 2; Crystal structure; Endogenic indole derivative; Complex
Evaluation of two anti-gp91phox antibodies as immunoprobes for Nox family proteins: mAb 54.1 recognizes recombinant full-length Nox2, Nox3 and the C-terminal domains of Nox1-4 and cross-reacts with GRP 58 by Danas Baniulis; Yoko Nakano; William M. Nauseef; Botond Banfi; Guangjie Cheng; David J. Lambeth; James B. Burritt; Ross M. Taylor; Algirdas J. Jesaitis (pp. 186-196).
Progress in the study of Nox protein expression has been impeded because of the paucity of immunological probes. The large subunit of human phagocyte flavocytochrome b558 (Cytb), gp91phox, is also the prototype member of the recently discovered family of NADPH oxidase (Nox) proteins. In this study, we have evaluated the use of two anti-gp91phox monoclonal antibodies, 54.1 and CL5, as immunoprobes for Nox family proteins. Sequence alignment of gp91phox with Nox1, Nox3 and Nox4 identified regions of the Nox proteins that correspond to the gp91phox epitopes recognized by mAb 54.1 and CL5. Antibody 54.1 produced positive immunoblots of recombinant C-terminal fragments of these homologous proteins expressed in E. coli. Furthermore, only mAb 54.1 recognized full-length murine and human Nox3 expressed in HEK-293 cells, in immunoblots of alkali-stripped or detergent-solubilized membranes. 54.1 recognized Nox3 expression-specific proteins with Mr 30,000, 50,000, 65,000 and 88,000 for the murine protein and Mr of 38,000–58,000, 90,000, 100,000–130,000 and a broad species of higher than 160,000 for the human protein. We conclude that mAb 54.1 can serve as a probe of Nox3 and possibly other Nox proteins, if precautions are taken to remove GRP 58 and other crossreactive membrane-associated or detergent-insoluble proteins from the sample to be probed.
Keywords: Abbreviations; 2-DE; two dimensional electrophoresis; ASB-14; amidosulfobetaine-14; ATP; adenosine 5′-triphosphate; BCIP/NBT; 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium; CHAPS; (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; CGD; chronic granulomatous disease; Cytb; flavocytochrome; b; 558; DEAE; diethylaminoethyl; DFP; diisopropyl fluorophosphate; DMEM; Dulbecco's modified Eagle's medium; DTT; dithiothreitol; EDTA; ethylenediaminetetraacetic acid; ER; endoplasmic reticulum; FAD; flavin adenine dinucleotide; FBS; fetal bovine serum; FITC; fluorescein isothiocyanate; HEPES; N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]; IEF; isoelectric focusing; IPTG; isopropyl β-; d; -1-thiogalactopyranoside; mAb; monoclonal antibody; MALDI-TOF; matrix-assisted, laser desorption/ionization-time-of-flight; Mr; molecular weight; MRB; membrane resuspension buffer (10 mM HEPES, 10 mM NaCl, 100 mM KCl, 1 mM EDTA, 0.1 mM dithiothreitol, 1 mM PMSF, 10 μg/ml chymostatin, pH-7.4); NADPH; β-nicotinamide adenine dinucleotidephosphate, reduced; PBS; phosphate buffered saline (10 mM phosphate, 150 mM NaCl, pH-7.4); PIPES; 1,4-piperazinediethanesulfonic acid; PMSF; phenylmethylsulfonyl fluoride; SDS; sodium dodecyl sulfate; SDS-PAGE; SDS polyacrylamide gel electrophoresis; Tris; tris(hydroxymethyl)aminomethane; YT; yeast extract/tryptone mediumNox; gp91; phox; Monoclonal antibody; Protein expression; Immunoblotting
Crystallization, X-ray diffraction analysis and phasing of carboxylesterase PA3859 from Pseudomonas aeruginosa by Alessandro Pesaresi; Doriano Lamba (pp. 197-201).
We have recently purified an intracellular carboxylesterase encoded by the open reading frame PA3859 of Pseudomonas aeruginosa. Among proteins showing a significant sequence homology with PA3859 the in vivo function is only known for the human acyl-protein thioesterase I that is involved in the deacylation of Gα proteins. The crystal structure determination of P. aeruginosa carboxylesterase is expected to provide insights into its physiological role. Therefore, the PA3859 gene was cloned and heterologously expressed in Escherichia coli as N-terminally 6xHis tagged recombinant protein. Here, we present the crystallization, X-ray diffraction analysis and phasing of this enzyme. Two crystal forms were obtained by the hanging drop vapor diffusion method. Crystals of form I belong to the space group P21 with cell dimensions of a=65.65, b=50.55, c=142.55 Å, β=92.9° and diffracted, upon flash annealing, up to a resolution of 2.9 Å. Two dimers are present in the asymmetric unit. Crystals of form II belong to space group P21212, with unit cell dimensions of a=96.42, b=96.36, c=68.04 Å and diffracted up to 2.1 Å resolution. One dimer is present in the asymmetric unit.
Keywords: Pseudomonas aeruginosa; Carboxylesterase; α/β hydrolase; Crystallization; X-ray crystal structure
Crystallization and preliminary X-ray crystallographic studies of Protac®, a commercial protein C activator isolated from Agkistrodon contortrix contortrix venom by Mário T. Murakami; Raghuvir K. Arni (pp. 202-204).
The protein C pathway plays an important role in the control and regulation of the blood coagulation cascade and prevents the propagation of the clotting process on the endothelium surface. In physiological systems, protein C activation is catalyzed by thrombin, which requires thrombomodulin as a cofactor. The protein C activator from Agkistrodon contortrix contortrix acts directly on the zymogen of protein C converting it into the active form, independently of thrombomodulin. Suitable crystals of the protein C activator from Agkistrodon contortrix contortrix were obtained from a solution containing 2 M ammonium sulfate as the precipitant and these crystals diffracted to 1.95 Å resolution at a synchrotron beamline. The crystalline array belongs to the monoclinic space group C2 with unit cell dimensions a=80.4, b=63.3 and c=48.2 Å, α= γ=90.0° and β=90.8°.
Keywords: Protein C activator; Serine proteinase; Agkistrodon contortrix contortrix; venom; X-ray diffraction analysis