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BBA - Proteins and Proteomics (v.1748, #2)
A common docking site for response regulators on the yeast phosphorelay protein YPD1 by Stace W. Porter; Ann H. West (pp. 138-145).
In Saccharomyces cerevisiae, a multi-component phosphorelay signal transduction pathway mediates cellular responses to environmental stress. A histidine-containing phosphotransfer protein, YPD1, represents a bifurcation point between the SLN1–YPD1–SSK1 pathway responsible for osmotic stress responses and the SLN1–YPD1–SKN7 pathway involved in cell wall biosynthesis and cell cycle control. The phosphorelay protein YPD1 must physically interact with and transfer phosphoryl groups between three homologous response regulator domains, designated SLN1-R1, SSK1-R2, and SKN7-R3. In this comparative study, the molecular basis of interaction was examined between YPD1 and each of the three response regulator domains utilizing alanine scanning mutagenesis combined with a yeast two-hybrid assay. Results from the yeast two-hybrid assay indicate that all three response regulator domains bind to a common area, largely hydrophobic in nature, on the surface of YPD1. We postulate that other YPD1 surface residues surrounding this common docking site are involved in making specific interactions with one or more of the response regulator domains.
Keywords: Abbreviations; MAP kinase; mitogen-activated protein kinase; HPt; histidine-containing phosphotransfer; CPRG; chlorophenolred β-; d; -galactopyranoside; SLN1-R1; response regulator domain of SLN1; SSK1-R2; response regulator domain of SSK1; SKN7-R3; response regulator domain of SKN7; PCR; polymerase chain reactionHistidine-containing phosphotransfer (HPt) protein; Two-component signal transduction; Protein–protein interaction; Yeast two-hybrid assay; Response regulator; Osmoregulation
N-terminal control of small heat shock protein oligomerization: changes in aggregate size and chaperone-like function by Cheryl Eifert; Michael R. Burgio; Pauline M. Bennett; John C. Salerno; Jane F. Koretz (pp. 146-156).
The small heat shock protein superfamily is composed of proteins from throughout the phylogenetic spectrum that are induced upon environmental stress. Their structural stability under stress derives in large part from the central region of the proteins, which forms two beta sheets held together by hydrophobic interactions and appears to be present in all superfamily members. The length, sequence, and amino acid composition of the N- and C-terminals, in contrast, are quite variable. The role of the N-terminal has been hypothesized to control species-specific assembly of subunits into higher level structures. To test this, a set of constructs was designed and expressed: the N-terminal sequences preceding the start of the core regions of αA-crystallin and HSP 16.5 from Methanococcus jannaschii were swapped; the N-terminal of each protein was removed, and replaced with a brief N-terminal extension sequence; and two nonsense N-terminal sequences of approximately the same length and hydropathicity as the original replaced the αA-crystallin N-terminal. All constructs, plus the original recombinant sequences, could be overexpressed except for the 16.5 N-terminal extension, and all showed chaperone-like activity except for the hybrid with the 16.5 C-terminal. Size and properties of the replacement N-terminal place limits on aggregate size. Additional restrictions are imposed by the structure of the dimer.
Keywords: Small heat shock protein; Alpha-crystallin; Mutagenesis; Subunit interaction; Quaternary structure; Chaperone-like activity
Calcium-induced conformational changes of the recombinant CBP3 protein from Dictyostelium discoideum by Tsogbadrakh Mishig-Ochiriin; Chang-Hun Lee; Sun-Young Jeong; Beom-Jun Kim; Chang-Hoon Choi; Hyung-Soon Yim; Sa-Ouk Kang (pp. 157-164).
Calcium-binding proteins play various and significant roles in biological systems. Conformational changes in their structures are closely related to their physiological functions. To understand the role of calcium-binding protein 3 (CBP3) in Dictyostelium discoideum, its recombinant proteins were analyzed using circular dichroism (CD) and fluorescence spectroscopy. Gel mobility shift analysis showed that Ca2+ induced a mobility shift of the recombinant CBP3. Far ultra-violet CD spectra and intrinsic fluorescence spectra on CBP3 and its N- and C-terminal domains exhibited that they underwent a conformational rearrangement depending upon Ca2+ binding. Measurement of Ca2+ dissociation constants demonstrated that CBP3 had high affinity toward Ca2+ in the sub-micromolar range and N-terminal domain had higher affinity than C-terminal domain. The changes of fluorescence spectra by an addition of 8-anilino-1-naphthalene sulfonic acid indicated that the hydrophobic patches of CBP3 and its C-terminal domain are likely to be more exposed in the presence of Ca2+. Since the exposure of hydrophobic patches is thermodynamically unfavorable, Ca2+-bound CBP3 may interact with other proteins in vivo. All these data suggest that Ca2+ induces CBP3 to be more favorable conformation to interact with target proteins.
Keywords: Calcium-binding protein 3; Conformational rearrangement; Dictyostelium discoideum
Probing actomyosin interactions with 2,4-dinitrophenol by A.S. Ribeiro; V.P. Salerno; M. Sorenson (pp. 165-173).
Access to different intermediates that follow ATP cleavage in the catalytic cycle of skeletal muscle actomyosin is a major goal of studies that aim toward an understanding of chemomechanical coupling in muscle contraction. 2,4-Dinitrophenol (DNP, 10−2 M) inhibits muscle contraction, even though it accelerates the ATPase activity of isolated myosin. Here we used myosin subfragment 1 (S1), acto-S1 and mammalian skinned fibers to investigate the action of DNP in the presence of actin. DNP increases acto-S1 affinity and at the same time reduces the maximum rate of turnover as [actin]→ ∞. In skinned fibers, isometric force is reduced to the same extent ( K0.5≅6 mM). Although actin activates Pi release from S1 at all DNP concentrations tested, the combination of enhanced S1 activity and reduced acto-S1 activity leads to a reduction in the ratio of these two rates by a factor of 30 at the highest DNP concentration tested. This effect is seen at low as well as at high actin concentrations and is less pronounced with the analog meta-nitrophenol (MNP), which does not inhibit the acto-S1 ATPase. Arrhenius plots for acto-S1 are parallel and linear between 5 and 30 °C, indicating no abrupt shifts in rate-limiting step with either DNP or MNP. Analysis of the reduction in isometric force with increasing Pi concentrations suggests that DNP and MNP stabilize weakly bound cross-bridges (AM.ADP.Pi). In addition, MNP (10−2 M) increases the apparent affinity for Pi.
Keywords: Acto-S1; Skinned fiber; Chemomechanical coupling; Pi binding; Cross-bridge; Nitrophenol
Comparative analysis of the cytotoxicity of homopolymeric amino acids by Yoko Oma; Yoshihiro Kino; Noboru Sasagawa; Shoichi Ishiura (pp. 174-179).
Many human proteins have homopolymeric amino acid (HPAA) tracts, although the physiological significance or cellular effects of their presence is poorly understood. We previously reported that 20 kinds of HPAAs show characteristic intracellular localization and that among those, hydrophobic HPAAs aggregate strongly and form high molecular weight proteins when expressed in cultured cells. In this study, we investigated the cytotoxicity of 20 kinds of HPAAs. HPAA tracts of ∼30 residues fused to the C-terminus of YFP were expressed in COS-7 cells. Cells expressing homopolymeric-Cys, -Ile, -Leu, and -Val showed low viability in Trypan Blue assay. Caspase-3 activity, which is usually upregulated in dying cells, was determined by measuring the cleavage of the peptide substrate Ac–DEVD–MCA and by detecting the cleaved active form of the caspase-3 by Western blotting. The activity of caspase-3 was drastically elevated in cells expressing those HPAAs which showed low viability in Trypan Blue assay. Interestingly, it was found that there is a correlation between the hydrophobicity of a single amino acid and the cytotoxicity of the corresponding HPAA as a homopolymer. These results indicate that the hydrophobicity of HPAAs may cause cytotoxicity.
Keywords: Polyglutamine; Polyalanine; Triplet repeat; Cell death; Cytotoxicity; Caspase
Overexpression and characterization of a novel chitinase from Trichoderma atroviride strain P1 by Ingunn A. Hoell; Sonja S. Klemsdal; Gustav Vaaje-Kolstad; Svein J. Horn; Vincent G.H. Eijsink (pp. 180-190).
We describe the overexpression and characterization of a new 30 kDa family 18 chitinase (Ech30) from Trichoderma atroviride strain P1. Sequence alignments indicate that the active site architecture of Ech30 resembles that of endochitinases such as hevamine from the rubber tree ( Hevea brasiliensis). The ech30 gene was overexpressed in Escherichia coli without its signal peptide and with an N-terminal His-tag. The enzyme was produced as inclusion bodies, from which active chitinase could be recovered using a simple refolding procedure. The enzyme displayed an acidic pH-optimum (pH 4.5–5.0), probably due to the presence of a conserved Asn residue near the catalytic glutamate, which is characteristic for acidic family 18 chitinases. Studies with oligomers of N-acetylglucosamine [(GlcNAc) n], 4-methylumbelliferyl (4-MU) labelled GlcNAc oligomers and β-chitin reveal enzymatic properties typical of an endochitinase: 1) low activity towards short substrates (kinetic parameters for the hydrolysis of 4-MU-(GlcNAc)2 were Km, 149+/−29 μM and kcat, 0.0048+/−0.0005 s-1), and 2) production of relatively large amounts of trimers and tetramers during degradation of β-chitin. Detailed studies with GlcNAc oligomers indicated that Ech30 has as many as seven subsites for sugar binding. As expected for a family 18 chitinase, catalysis proceeded with retention of the β-anomeric configuration.
Keywords: Chitin; Chitinase; Chitooligosaccharides; Ech30; Glycoside hydrolase; Trichoderma
Proteomic analysis of Bacillus anthracis Sterne vegetative cells by Anthony W. Francis; Christy E. Ruggiero; Andrew T. Koppisch; Jingao Dong; Jian Song; Thomas Brettin; Srinivas Iyer (pp. 191-200).
Mass spectrometry and proteomics have found increasing use as tools for the rapid detection of pathogenic bacteria, even when they are in a mixture of non-pathogenic relatives. The success of this technique is greatly augmented by the availability of publicly accessible proteomic databases for specific pathogenic bacteria. To aid proteomic detection analyses for the causative agent of anthrax, we have constructed a comprehensive proteomic catalogue of vegetative Bacillus anthracis Sterne cells using liquid chromatography tandem-mass spectrometry. Proteins were separated by molecular weight or isoelectric point prior to tryptic digestion. Alternatively, the whole protein extract was digested and tryptic peptides were separated by cation exchange chromatography prior to Reverse Phase-LC-MS/MS. The use of three complementary, pre-analytical separation techniques resulted in the identification of 1048 unique proteins, including 694 cytosolic, 153 membrane (including 27 cell wall), and 30 secreted proteins, accounting for 19% of the total predicted proteome. Each identified protein was functionally categorized using the gene attribute database from TIGR CMR. These results provide a large proteomic catalogue of vegetative B. anthracis cells and, coupled with the recent proteomic catalogue of B. anthracis spore proteins, form a thorough summary of proteins expressed in the active and dormant stages of this organism.
Keywords: Bacillus anthracis; Sterne; Proteomics; Mass spectrometry
The structure of Apo R268A human aldose reductase: Hinges and latches that control the kinetic mechanism by Kurt M. Bohren; June M. Brownlee; Amy C. Milne; Kenneth H. Gabbay; David H.T. Harrison (pp. 201-212).
Aldose reductase (AR) catalyzes the NADPH-dependent reduction of glucose and other sugars to their respective sugar alcohols. The NADP+/NADPH exchange is the rate-limiting step for this enzyme and contributes in varying degrees to the catalytic rates of other aldo–keto reductase superfamily enzymes. The mutation of Arg268 to alanine in human recombinant AR removes one of the ligands of the C2-phosphate of NADP+ and markedly reduces the interaction of the apoenzyme with the nucleotide. The crystal structure of human R268A apo-aldose reductase determined to a resolution of 2.1 Å is described. The R268A mutant enzyme has similar kinetic parameters to the wild-type enzyme for aldehyde substrates, yet has greatly reduced affinity for the nucleotide substrate which greatly facilitates its crystallization in the apoenzyme form. The apo-structure shows that a high temperature factor loop (between residues 214 and 226) is displaced by as much as 17 Å in a rigid body fashion about Gly213 and Ser226 in the absence of the nucleotide cofactor as compared to the wild-type holoenzyme structure. Several factors act to stabilize the NADPH-holding loop in either the ‘open’ or ‘closed’ conformations: (1) the presence and interactions of the nucleotide cofactor, (2) the residues surrounding the Gly213 and Ser226 hinges which form unique hydrogen bonds in the ‘open’ or ‘closed’ structure, and (3) the Trp219 “latch� residue which interacts with an arginine residue, Arg293, in the ‘open’ conformation or with a cysteine residue, Cys298, in the ‘closed’ conformation. Several mutations in and around the high temperature factor loop are examined to elucidate the role of the loop in the mechanism by which aldose reductase binds and releases its nucleotide substrate.
Keywords: Abbreviations; NADP; +; nicotinamide adenosine diphosphate 2′-phosphate (oxidized); NADPH; nicotinamide adenosine diphosphate 2′-phosphate (reduced); AKR; aldo–keto reductaseAldose reductase; Apo structure; Loop movement; X-ray structure; Enzyme kinetics
The Saccharomyces cerevisiae ORF YNR064c protein has characteristics of an ‘orphaned’ epoxide hydrolase by Lisa T. Elfström; Mikael Widersten (pp. 213-221).
The open reading frame YNR064c in Saccharomyces cerevisiae encodes a protein tentatively assigned as similar to a bacterial dehalogenase. In this study we conclude that the YNR064c protein displays characteristics of an epoxide hydrolase belonging to the α/β-hydrolase fold family of enzymes. Endogenous expression of the protein in S. cerevisiae was confirmed and a His-tagged variant of the protein was heterologously expressed in both Escherichia coli and Pichia pastoris for isolation and characterization. The YNR064c protein displayed low but reproducible epoxide hydrolase activity with racemic phenanthrene 9,10-oxide and trans- or cis-stilbene oxide. Phylogenetic analysis of related gene products found in various microorganisms suggested that the YNR064c protein is a member of a new subclass of α/β-hydrolase fold enzymes.
Keywords: Abbreviations; EH; epoxide hydrolase; ORF; open reading frameUnidentified ORF; α/β-hydrolase fold; Saccharomyces cerevisiae; Heterologous expression
Heterologous expression, purification, crystallization, X-ray analysis and phasing of the acetyl xylan esterase from Bacillus pumilus by Ivet Krastanova; Corrado Guarnaccia; Sotir Zahariev; Giuliano Degrassi; Doriano Lamba (pp. 222-230).
Bacillus pumilus PS213 acetyl xylan esterase (AXE) acts as an accessory enzyme in the plant cell wall hemicellulose biodegradation pathway. It belongs to the carbohydrate esterase family 7 and hydrolyses the ester linkages of the acetyl groups in position 2 and/or 3 of the xylose moieties of the acetylated xylan fragments from hardwood. The enzyme displays activity towards a broad range of acetylated compounds including the antibiotic cephalosporin-C. In this study we report the heterologous expression, purification, physicochemical characterization and crystallization of the recombinant B. pumilus AXE. Remarkable improvement of the crystal quality was achieved by setting up crystallization conditions, at first established using the hanging drop vapor diffusion method, in a micro-batch experiment. Rod-like diffraction quality crystals were obtained using 10% PEG 6000, 0.1 M MES pH 6.0 and a wide range of LiCl concentrations (0.2–1.0 M) as precipitant agent. Two different crystal forms, both belonging to space group P21, were characterized, diffracting X-rays to 2.5 and 1.9 Å resolution. Successful molecular replacement showed 12 molecules in the asymmetric unit of either crystal forms that are arranged as two doughnut-like hexamers, each one encompassing a local 32 symmetry. A catalytic inactive mutant Ser181Ala of B. pumilus AXE was also engineered, expressed, purified and crystallized for functional and structural studies.
Keywords: Bacillus pumilus; Acetyl xylan esterase; Carbohydrate esterase CE7; α/β hydrolase; Micro-batch crystallization; X-ray crystal structure