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BBA - Proteins and Proteomics (v.1749, #1)
A new insight into mercurized hemoglobin aggregation mechanism by Anatoli E. Myshkin; Vera S. Khromova (pp. 1-6).
Coagulation of bovine oxyhemoglobin in the presence of mercuric acetate in concentrations within a range including concentrations exceeding those required to block the single pair of thiol groups of the protein has been investigated in Tris–acetate buffer. The values of initial coagulation rate plotted against mercury-to-hemoglobin molar ratio give curves exhibiting a clear break points at ratios corresponding to full blocking of the mentioned thiol groups. Larger amounts of mercury reagents producing enhanced protein coagulation effect depend approximately quadratically on the mercury concentration . Interaction of the excess mercuric ions with some mercury-binding sites located on or near the dimer–dimer contact surfaces of the protein producing stronger coagulation effect is suggested.
Keywords: Oxyhemoglobin; Aggregation; Mercury(II) ions; Coordination bonds
Biochemical characterization of recombinant acetyl xylan esterase from Aspergillus awamori expressed in Pichia pastoris: Mutational analysis of catalytic residues by Takuya Koseki; Yozo Miwa; Shinya Fushinobu; Katsumi Hashizume (pp. 7-13).
We engineered an acetyl xylan esterase ( AwaxeA) gene from Aspergillus awamori into a heterologous expression system in Pichia pastoris. Purified recombinant AwAXEA (rAwAXEA) displayed the greatest hydrolytic activity toward α-naphthylacetate (C2), lower activity toward α-naphthylpropionate (C3) and no detectable activity toward acyl-chain substrates containing four or more carbon atoms. Putative catalytic residues, Ser119, Ser146, Asp168 and Asp202, were substituted for alanine by site-directed mutagenesis. The biochemical properties and kinetic parameters of the four mutant enzymes were examined. The S119A and D202A mutant enzymes were catalytically inactive, whereas S146A and D168A mutants displayed significant hydrolytic activity. These observations indicate that Ser119 and Asp202 are important for catalysis. The S146A mutant enzyme showed lower specific activity toward the C2 substrate and higher thermal stability than wild-type enzyme. The lower activity of S146A was due to a combination of increased Km and decreased kcat. The catalytic efficiency of S146A was 41% lower than that of wild-type enzyme. The synthesis of ethyl acetate was >10-fold than that of ethyl n-hexanoate synthesis for the wild-type, S146A and D168A mutant enzymes. However, the D202A showed greater synthetic activity of ethyl n-hexanoate as compared with the wild-type and other mutants.
Keywords: Site-directed mutagenesis; Catalytic residue; Acetyl xylan esterase; Aspergillus awamori; Pichia pastoris
The role of tryptophan residues in the autoprocessing of prosubtilisin E by Michio Sone; Liliana Falzon; Masayori Inouye (pp. 15-22).
Subtilisin E, a serine protease from Bacillus subtilis, requires an N-terminal propeptide for its correct folding. The propeptide is autocleaved and digested by the subtilisin domain upon proper folding. Here we investigated the individual roles of the three Trp residues within the subtilisin domain (Trp106, Trp113 and Trp241) on propeptide processing, enzymatic activity and stability of subtilisin. When the propeptide processing was examined by SDS-PAGE after refolding by rapid dilution, the mutation at either position Trp106 or Trp113 was found to significantly delay the propeptide processing, while the mutation at Trp241 had no effect. Far-UV circular dichroism (CD) spectra of the mutants revealed that the mutations at the three positions did not affect appreciably the α-helix content of subtilisin. Secondary structure thermal unfolding monitored by CD spectroscopy revealed that none of the tryptophan residues had any significant effect on the stability of mature subtilisin. The enzymatic activity measurements showed that only Trp106 plays a major role in the enzymatic activity of subtilisin E. These results demonstrate that both Trp106 and Trp113 play a specific role in propeptide processing and enzymatic activity, while Trp241 plays no considerable role on any of these activities.
Keywords: Abbreviations; IMC; intramolecular chaperon; GdnHCl; guanidine hydrochloride; PAGE; polyacrylamide gel electrophoresisSubtilisin; Autoprocessing; Protein folding; Serine protease; Intramolecular chaperone; Propeptide
Proteome analysis of rat pancreas induced by pancreatectomy by Jun-Seop Shin; Jae-Jeong Lee; Eun-Ju Lee; Yeon-Hee Kim; Kwon-Seok Chae; Chan-Wha Kim (pp. 23-32).
The previous study demonstrated that the streptozotocin (STZ)-induced diabetic mice can be cured by injecting the regenerating pancreatic extract (RPE) of the partially pancreatectomized Wistar–Kyoto rats. In this study, to characterize the complex pattern of protein expression in RPE, the proteins of altered expression level after the pancreatectomy were identified by 2-dimensional electrophoresis (2-DE) and mass spectrometry. Of 76 significantly up- or down-regulated protein spots, 61 were identified by MALDI-TOF/MS. Moreover, the whole RPE was fractionated into 4 groups using an anion-exchange chromatography and each fraction's cell proliferating activity was measured by MTT assay. Compared to the normal pancreatic extract, fraction 3 and 4 of RPE showed the maximal cell proliferating activity. On 2-DE of 3 and 4 fractions, a total of 10 spots, which are differentially expressed after the pancreatectomy, were identified by MS/MS. Of these identified proteins, Reg III which might be functionally associated with well known regenerating factor (Reg I) was found. Taken together, our results demonstrated that the differential protein expression associated with pancreas regeneration could be sought by 2-DE and mass spectroscopy and suggested that the pre-fractionation method combined with in vitro cell proliferation assay is effectively used to pinpoint the active components for pancreas regeneration.
Keywords: Abbreviations; 2-DE; 2-dimensional electrophoresis; RPE; regenerating pancreatic extract; STZ; streptozotocin; MS; mass spectrometry; PBS; phosphate buffered saline; FBS; fetal bovine serum; MALDI-TOF; matrix-assisted laser-desorption/ionization time of flight; MTT; C; ,; N; -diphenyl-N′-4,5-dimethyl thiazol 2 yl tetrazolium bromide; MS/MS; tandem mass spectrometry; FPLC; fast protein liquid chromatographyPancreatectomy; Streptozotocin; Regeneration; Regenerating pancreatic extract; Reg III
Ionisations within a subtilisin–glyoxal inhibitor complex by Aleksandra Djurdjevic-Pahl; Chandralal Hewage; J. Paul G. Malthouse (pp. 33-41).
Z-Ala-Pro-Phe-glyoxal (where Z is benzyloxycarbonyl) has been shown to be a competitive inhibitor of subtilisin with a Ki=2.3±0.2 μM at pH 7.0 and 25 °C. Using Z-Ala-Pro-[2-13C]Phe-glyoxal we have detected a signal at 107.3 ppm by13C NMR, which we assign to the tetrahedral adduct formed between the hydroxy group of serine-195 and the13C-enriched keto-carbon of the inhibitor. The chemical shift of this signal is pH independent from pH 4.2 to 7.0 and we conclude that the oxyanion p Ka<3. This is the first observation of oxyanion formation in a reversible subtilisin–inhibitor complex. The inhibitor is bound as a hemiketal which is in slow exchange with the free inhibitor. Inhibitor binding depends on a p Ka of ∼6.5 in the free enzyme and on a p Ka<3.0 when the inhibitor is bound to subtilisin. Protonation of the oxyanion promotes the disassociation of the inhibitor. We show that oxyanion formation cannot be rate limiting during catalysis and that subtilisin stabilises the oxyanion by at least 45.1 kJ mol−1. We conclude that if the energy required for oxyanion stabilisation is utilised as binding energy in drug design it should make a significant contribution to inhibitor potency.
Keywords: Abbreviations; Z; benzyloxycarbonylOxyanion; p; K; a; Protease; Tetrahedral intermediate
An electrochemical investigation of hemoglobin and catalase incorporated in collagen films by Min Li; Pingli He; Yan Zhang; Naifei Hu (pp. 43-51).
Collagen, an electrochemically inert protein, formed films on pyrolytic graphite (PG) electrodes, which provided a suitable microenvironment for heme proteins to transfer electron directly with the underlying electrodes. Hemoglobin (Hb) and catalase (Cat) incorporated in collagen films exhibited a pair of well-defined and quasi-reversible cyclic voltammetric peaks at around −0.35 V and −0.47 V (vs. SCE) in pH 7.0 buffers, respectively, characteristic of the protein heme Fe(III)/Fe(II) redox couples. UV–vis spectra showed that the heme proteins in collagen films retained their near-native conformations in the medium pH range. The results of scanning electron microscopy (SEM) demonstrated that the interaction between heme proteins and collagen made the morphology of dry protein-collagen films different from the collagen films alone. The electrochemical parameters such as apparent heterogeneous electron transfer rate constant ( ks) and formal potential ( E°′) of the films were estimated by using square wave voltammograms (SWV) and nonlinear regression analysis. The heme protein-collagen film electrodes were also used to catalyze the reduction of nitrite, oxygen and hydrogen peroxide, indicating potential applications of the films for the fabrication of a new type of biosensor that does not use mediators.
Keywords: Hemoglobin; Catalase; Collagen; Direct electrochemistry; Electrochemical catalysis
Action pattern of Fusarium moniliforme endopolygalacturonase towards pectin fragments: Comprehension and prediction by G. André-Leroux; D. Tessier; E. Bonnin (pp. 53-64).
The structures of complexes of Fusarium moniliforme endopolygalacturonase (endoPG) with non-methylated or partly methylated homogalacturonan fragments were modeled to identify the residues involved in substrate binding and to correlate the cleavage pattern with the experimental productive modes. The conformational space of the complex was extensively explored and malto- to hexo-oligogalacturonates were modeled in the active cleft. To select the most highly probable productive complex for each oligomer between DP2 and 6, four energetic criteria were defined. Noteworthingly, the results were in accordance with the experimental results showing the mode of action of this enzyme towards un-methyl-esterified oligogalacturonates. Furthermore, the amino-acid residues involved in the binding were confirmed by similar studies performed on other endoPGs. Then, the oligomers were gradually methyl-esterified at one or more positions and similar docking experiments were carried out. Markedly, the docking energies were not significantly modified by the methyl-esterification of the substrate and it is likely that the methyl-esterification of the substrate does not alter the mode of action of the enzyme. Finally, 1D sequence and 3D structure of the endopolygalacturonase of Aspergillus niger II, known to be strictly non-tolerant to methylesters, were compared with the sequence and structure of the tolerant F. moniliforme endopolygalacturonase to get to a structural comprehension of the tolerant–or not–behaviour of endoPGs with methyl-esterified pectins.
Keywords: Endopolygalacturonase; Homogalacturonan; Action pattern; Molecular modeling; Subsite binding
Stochastic boundary molecular dynamics simulation ofl-ribose in the active site of Actinoplanes missouriensis xylose isomerase and its Val135Asn mutant with improved reaction rate by Harri Santa; Juha Kammonen; Olli Lehtonen; Johanna Karimäki; Ossi Pastinen; Matti Leisola; Ossi Turunen (pp. 65-73).
We used molecular dynamics simulations to study how a non-natural substrate,l-ribose, interacts with the active site of Actinoplanes missouriensis xylose isomerase. The simulations showed thatl-ribose does not stay liganded in the active site in the same way asd-xylose, in which the oxygens O2 and O4 are liganded to the metal M1. The oxygen O4 ofl-ribose moved away from the metal M1 to an upside down position. Furthermore, the distances of the carbons C1 and C2 ofl-ribose to the catalytic metal M2 were higher than in the case ofd-xylose. These findings explain the extremely low reaction rate of xylose isomerase withl-ribose. The mutation V135N close to the C5–OH of the substrate increased the reaction efficiency 2- to 4-fold withl-ribose. V135N did not affect the reaction withd-xylose andl-arabinose, whereas the reaction withd-glucose was impaired, probably due to a hydrogen bond between Asn-135 and the substrate. Whenl-ribose was the substrate, Asn-135 formed a hydrogen bond to Glu-181. As a consequence, O4 ofl-ribose stayed liganded to the metal M1 in the V135N mutant in molecular dynamics simulations. This explains the decreased Km of the V135N mutant withl-ribose.
Keywords: Xylose isomerase; l; -Ribose; Molecular dynamics; Site-directed mutagenesis; Catalytic efficiency
Characterization and study of a κ-casein-like chymosin-sensitive linkage by Isabelle Callebaut; Françoise Schoentgen; Karine Prat; Jean-Paul Mornon; Pierre Jollès (pp. 75-80).
The present report is dealing with the identification, in various unrelated proteins, of protein fragments sharing local sequence and structure similarities with the chymosin-sensitive linkage surrounding the Phe-Met/Ile bond of κ-caseins. In all these proteins, this linkage is observed within an exposed β-strand-like structure, as also predicted for κ-caseins. The structure of one of these fragments, included in glutamine synthetase, particularly superimposes well with the conformation observed for a chymosin inhibitor (CP-113972) within the complex it forms with chymosin and can be similarly accommodated by specificity pockets within the enzyme substrate binding cleft. The effect of the enzyme activity of chymosin was thus tested on glutamine synthetase. Chymosin cut the latter at the Phe-Met linkage, suggesting that this system may locally resemble the κ-casein/chymosin complex.
Keywords: Kappa-casein; Chymosin; Glutamine synthetase; Secondary structure; Enzyme plasticity
Apolipoprotein AI could be a significant determinant of epithelial integrity in rainbow trout gill cell cultures: A study in functional proteomics by Richard W. Smith; Chris M. Wood; Phil Cash; Linda Diao; Peter Pärt (pp. 81-93).
The freshwater fish gill forms a barrier against an external hypotonic environment. By culturing rainbow trout gill cells on permeable supports, as intact epithelia, this study investigates barrier property mechanisms. Under symmetrical conditions the apical and basolateral epithelial surfaces contact cell culture media. Replacing apical media with water, to generate asymmetrical conditions (i.e. the situation encountered by the freshwater gill), rapidly increases transepithelial resistance (TER). Proteomic analysis revealed that this is associated with enhanced expression of pre-apolipoprotein AI (pre-apoAI). To test the physiological relevance, gill cells were treated with a dose of 50 μg ml−1 human apolipoprotein (apoAI). This was found to elevate TER in those epithelia which displayed a lower TER prior to apoAI treatment. These results demonstrate the action of apoAI and provide evidence that the rainbow trout gill may be a site of apoAI synthesis. TER does not differentiate between the trans-cellular (via the cell membrane) and para-cellular (via intercellular tight junctions) pathways. However, despite the apoAI-induced changes in TER, para-cellular permeability (measured by polyethylene glycol efflux) remained unaltered suggesting apoAI specifically reduces trans-cellular permeability. This investigation combines proteomics with functional measurements to show how a proteome change may be associated with freshwater gill function.
Keywords: Proteome; Apolipoprotein AI; Transepithelial resistance; Trans-cellular flux
Detection of activity and mass spectrometric identification of mouse liver carboxylesterase and aldehyde dehydrogenase separated by non-denaturing two-dimensional electrophoresis after extraction with detergents by Youji Shimazaki; Takashi Manabe (pp. 95-101).
To examine the activities and identity of enzymes associated with organelles such as microsomes and mitochondria, proteins from mouse liver were extracted using the non-ionic detergents Nonidet P-40 (NP-40), polyoxyethylene sorbitan monooleate (Tween 80), polyoxyethylene isooctylphenyl ester (Triton X), n-octyl β-d-glucoside (octyl glycoside) or anionic detergent sodium dodecylsulfate (SDS) after the removal of cytosolic proteins. The proteins extracted by detergents were separated by non-denaturing two-dimensional electrophoresis (2-DE). The activities of esterase and aldehyde dehydrogenase were retained by non-denaturing 2-DE after treatment with each non-ionic detergent, but the activities were reduced or lost when the proteins were extracted with more than 0.5% SDS. For proteomic analysis of the organelle-associated proteins in mouse liver, proteins were separated by non-denaturing 2-DE and were identified using electrospray ionization tandem mass spectrometry (ESI-MS/MS) after the proteins were solubilized by octyl glycoside, NP-40 and 0.1% SDS. Several organelle-associated proteins such as carboxylesterase, aldehyde dehydrogenase, glucose regulated protein and HSP60 were identified. These results indicate that the activities and identity of detergent-soluble enzymes can be examined by this non-denaturing 2-DE and mass spectrometry.
Keywords: Abbreviations; Tween 80; polyoxyethylene sorbitan monooleate; Triton X; polyoxyethylene isooctylphenyl esterEnzyme activity; Non-denaturing two-dimensional electrophoresis; Electrospray ionization tandem mass spectrometry; Non-ionic detergent; Sodium dodecylsulfate
Two consecutive aspartic acid residues conferring herbicide resistance in tobacco acetohydroxy acid synthase by Dung Tien Le; Moon-Young Yoon; Young Tae Kim; Jung-Do Choi (pp. 103-112).
Acetohydroxy acid synthase (AHAS) catalyzes the first common step in the biosynthesis pathway of the branch chain amino acids in plants and microorganisms. A great deal of interest has been focused on AHAS since it was identified as the target of several classes of potent herbicides. In an effort to produce a mutant usable in the development of an herbicide-resistant transgenic plant, two consecutive aspartic acid residues, which are very likely positioned next to the enzyme-bound herbicide sulfonylurea as the homologous residues in AHAS from yeast, were selected for this study. Four single-point mutants and two double mutants were constructed, and designated D374A, D374E, D375A, D375E, D374A/D375A, and D374E/D375E. All mutants were active, but the D374A mutant exhibited substrate inhibition at high concentrations. The D374E mutant also evidenced a profound reduction with regard to catalytic efficiency. The mutation of D375A increased the Km value for pyruvate nearly 10-fold. In contrast, the D375E mutant reduced this value by more than 3-fold. The double mutants exhibited synergistic reduction in catalytic efficiencies. All mutants constructed in this study proved to be strongly resistant to the herbicide sulfonylurea Londax. The double mutants and the mutants with the D375 residue were also strongly cross-resistant to the herbicide triazolopyrimidine TP. However, only the D374A mutant proved to be strongly resistant to imidazolinone Cadre. The data presented here indicate that the two residues, D374 and D375, are located at a common binding site for the herbicides sulfonylurea and triazolopyrimidine. D375E may be a valuable mutant for the development of herbicide-resistant transgenic plants.
Keywords: Abbreviations; AHAS; Acetohydroxy acid synthase; mAHAS; Mutant AHAS; wAHAS; Wild type AHAS; CD; Circular dichroism; FAD; Flavine adenine dinucleotide; GSH; Glutathione; GST; Glutathione; S; -transferase; IPTG; Isopropyl-β-; d; -thiogalactoside; PCR; Polymerase chain reaction; pGEX–AHAS; Plasmid derived from pGEX-2T containing tobacco AHAS cDNA; ThDP; Thiamine diphosphate; TP; Triazolopyrimidine sulfonamideAcetohydroxy acid synthase; Conserved aspartic acid; Herbicide resistance; Tobacco; Site-directed mutagenesis
Analysis of ligation and DNA binding by Escherichia coli DNA ligase (LigA) by Adam Wilkinson; Andrew Smith; Desmond Bullard; Manuel Lavesa-Curto; Heather Sayer; Alexandra Bonner; Andrew Hemmings; Richard Bowater (pp. 113-122).
NAD+-dependent DNA ligases are essential enzymes in bacteria, with the most widely studied of this class of enzymes being LigA from Escherichia coli. NAD+-dependent DNA ligases comprise several discrete structural domains, including a BRCT domain at the C-terminus that is highly-conserved in this group of proteins. The over-expression and purification of various fragments of E. coli LigA allowed the investigation of the different domains in DNA-binding and ligation by this enzyme. Compared to the full-length protein, the deletion of the BRCT domain from LigA reduced in vitro ligation activity by 3-fold and also reduced DNA binding. Using an E. coli strain harbouring a temperature-sensitive mutation of ligA, the over-expression of protein with its BRCT domain deleted enabled growth at the non-permissive temperature. In gel-mobility shift experiments, the isolated BRCT domain bound DNA in a stable manner and to a wider range of DNA molecules compared to full LigA. Thus, the BRCT domain of E. coli LigA can bind DNA, but it is not essential for DNA nick-joining activity in vitro or in vivo.
Keywords: BRCT domain; DNA ligase; Ligation; NAD; +; Protein–DNA binding
Mutation analysis of the human 5-lipoxygenase C-terminus: Support for a stabilizing C-terminal loop by Hisayo Okamoto; Tove Hammarberg; Ying-Yi Zhang; Bengt Persson; Takashi Watanabe; Bengt Samuelsson; Olof Rådmark (pp. 123-131).
Lipoxygenases contain prosthetic iron, in human 5-lipoxygenase (5LO) the C-terminal isoleucine carboxylate constitutes one of five identified ligands. ATP is one of several factors determining 5LO activity. We compared properties of a series of 5LO C-terminal deletion mutants (one to six amino acid residues deleted). All mutants were enzymatically inactive (expected due to loss of iron), but expression yield (in E. coli) and affinity to ATP–agarose was markedly different. Deletion of up to four C-terminal residues was compatible with good expression and retained affinity to the ATP-column, as for wild-type 5LO. However when also the fifth residue was deleted (Asn-669) expression yield decreased and the affinity to ATP was markedly diminished. This was interpreted as a result of deranged structure and stability, due to loss of a hydrogen bond between Asn-669 and His-399. Mutagenesis of these residues supported this conclusion. In the structure of soybean lipoxygenase-1, a C-terminal loop was pointed out as important for correct orientation of the C-terminus. Accordingly, a hydrogen bond appears to stabilize such a C-terminal loop also in 5LO.
Keywords: Abbreviations; 5H(P)ETE; 5(S)-hydro(pero)xy-6-trans-8,11,14-cis-eicosatetraenoic acid; 5LO; 5-lipoxygenase; SLO-1; soybean lipoxyganese-15-Lipoxygenase; Lipoxygenase; Leukotriene; Arachidonic acid; Mutagenesis
Proteomic analysis of complexes formed by human topoisomerase I by Alicja Czubaty; Agnieszka Girstun; Barbara Kowalska-Loth; Agata M. Trzcińska; Elżbieta Purta; Alicja Winczura; Wojciech Grajkowski; Krzysztof Staroń (pp. 133-141).
Human topoisomerase I is a nuclear enzyme that catalyses DNA relaxation and phosphorylation of SR proteins. Topoisomerase I participates in several protein–protein interactions. We performed a proteomic analysis of protein partners of topoisomerase I. Two methods were applied to proteins of the nuclear extract of HeLa cells: a co-immunoprecipitation and an affinity chromatography combined with mass spectrometry. Complexes formed by topoisomerase I with its protein partners were immunoprecipitated by scleroderma anti-topoisomerase I antibodies. To identify binding sites for the protein partners, baits corresponding to fragments of topoisomerase I were constructed and used in the affinity chromatography. The N-terminal domain and the cap region of the core domain appeared to be the main regions that bound proteins. We identified 36 nuclear proteins that were associated with topoisomerase I. The proteins were mainly involved in RNA metabolism. We found 29 new and confirmed 7 previously identified protein partners of topoisomerase I. More than 40% proteins that associate with the cap region contain two closely spaced RRM domains. Docking calculations identified the RRM domains as a possible site for the interaction of these proteins with the cap region.
Keywords: Proteomics; Topoisomerase I; Protein partners; Co-immunoprecipitation; Affinity chromatography
Crystallization of Halorhodopsin from Halobacterium sp. shark by Hirokazu Nishida; Takeshi Sakamoto; Tomoko Takeshita; Jun Otomo (pp. 143-145).
The chloride-ion-pumping channel, halorhodopsin from Halobacterium sp. shark was detergent-solubilized and 3-D crystallized. Proteins were solubilized using the nonionic detergent n-octyl-β-d-glucoside and were crystallized as thin-plate crystals with polyethylene glycol 4000 as a precipitant. The crystals belong to the space group P41212 with unit-cell dimensions a= b=74.5 Å and c=138.6 Å. The diffraction pattern was slightly anisotropic. The best ordered crystal diffracted up to 3.3 Å resolution along c axis with synchrotron radiation.
Keywords: Crystallization; Membrane protein; Rhodopsin; Halorhodopsin