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BBA - Proteins and Proteomics (v.1752, #1)

Editorial Board (pp. ii).

The protein composition of honeybee venom reconsidered by a proteomic approach by Nico Peiren; Frank Vanrobaeys; Dirk C. de Graaf; Bart Devreese; Jozef Van Beeumen; Frans J. Jacobs (pp. 1-5).

Pure honeybee venom samples were submitted to two-dimensional gel electrophoresis. A total of 49 excised spots were analyzed by mass spectrometry; 39 of them resulted in the identification of 6 different known bee venom proteins and of 3 proteins that have not been described in such samples before. The first new venom protein has a platelet-derived and vascular endothelial growth factor family domain, the second protein shows no homologies with any known protein and the third matches a hypothetical protein similar to major royal jelly protein 8.

Keywords: Abbreviations; 2-DE; two-dimensional gel electrophoresis; ESI; electrospray ionization; EST; expressed sequence tag; IEF; isoelectric focusing; IPG; immobilized pH gradient; IUIS; International Union of Immunology Societies; LC; liquid chromatography; MALDI; matrix-assisted laser desorption ionization; MRJP; major royal jelly protein; MS; mass spectrometry; PDGF/VEGF; platelet-derived and vascular endothelial growth factor; PLA2; phospholipase A2; TOF; time of flightHoneybee; Hymenoptera; Venom; Proteome analysis; Two-dimensional gel electrophoresis

High-throughput solubility assay for purified recombinant protein immunogens by Maria Stenvall; Johanna Steen; Mathias Uhl�n; Sophia Hober; Jenny Ottosson (pp. 6-10).

A high-throughput assay is described for analysis of the solubility of purified recombinant proteins. The assay is based on affinity purification of proteins in the presence of chaotropic agents followed by a dilution and incubation step to investigate the solubility in the absence of high concentrations of such agents. The assay can be performed in a 96-well format, which makes it well suited for high-throughput applications. For 125 recombinant proteins expressed as part of an antibody-based proteomics effort, experimental solubility data were compared to calculated hydrophobicity values based on the amino acid sequence of each protein. This comparison showed only weak correlation between the theoretical and experimental values, which emphasizes the importance of experimental assays to determine the solubility of recombinant proteins.

Keywords: Protein solubility; High-throughput; In vitro assay; Urea

Opposite roles of domains 2+3 of Escherichia coli EF-Tu and Bacillus stearothermophilus EF-Tu in the regulation of EF-Tu GTPase activity by Hana Šanderová; Jiří Jonák (pp. 11-17).

The effect of noncatalytic domains 2+3 on the intrinsic activity and thermostability of the EF-Tu GTPase center was evaluated in experiments with isolated domains 1 and six chimeric variants of mesophilic Escherichia coli (Ec) and thermophilic Bacillus stearothermophilus (Bst) EF-Tus. The isolated catalytic domains 1 of both EF-Tus displayed similar GTPase activities at their optimal temperatures. However, noncatalytic domains 2+3 of the EF-Tus influenced the GTPase activity of domains 1 differently, depending on the domain origin. Ecdomains 2+3 suppressed the GTPase activity of the Ecdomain 1, whereas those of BstEF-Tu stimulated the Bstdomain 1 GTPase. Domain 1 and domains 2+3 of both EF-Tus positively cooperated to heat-stabilize their GTPase centers to attain optimal activity at a temperature close to the optimal growth temperature of either organism. This can be explained by a stabilization effect of domains 2+3 on α-helical regions of the G-domain as revealed by CD spectroscopy.

Keywords: Abbreviations; EF-Tu; elongation factor Tu; Ec; Escherichia coli; Bst; Bacillus stearothermophilusElongation factor Tu; G-domain; GTPase regulation; Thermostability; Chimeric EF-Tu

Elevated expression temperature in a mesophilic host results in increased secretion of a hyperthermophilic enzyme and decreased cell stress by Jason D. Smith; Nicole E. Richardson; Anne S. Robinson (pp. 18-25).

Efficient protein folding and trafficking are essential for high-level production of secretory proteins. Slow folding or misfolding of proteins can lead to secretory bottlenecks that reduce productivity. We previously examined the expression of a hyperthermophilic tetramer Pyrococcus furiosus β-glucosidase in the yeast Saccharomyces cerevisiae. A secretory bottleneck was found in the endoplasmic reticulum, presumably due to β-glucosidase misfolding. By increasing expression temperature from 30 °C up to 40 °C, secretion yields increased by as much as 440% per cell to greater than 100 mg/L at 37 °C. We examined the effect of temperature on β-glucosidase folding and secretion and determined that increased expression temperature decreased intracellularly retained, insoluble β-glucosidase. Likewise, stress on the cell caused by β-glucosidase expression was found to be greatly reduced at 37 °C compared to 30 °C. Levels of the abundant endoplasmic reticulum chaperone, BiP, were relatively unchanged at these temperatures during heterologous expression. Using cycloheximide to inhibit new protein synthesis, we determined that the increase in secretion is likely due to the effect of temperature on the β-glucosidase itself rather than the cell's response to elevated temperatures. We believe that this is the first evidence of in vivo effects of temperature on the secretion of hyperthermophilic proteins.

Keywords: Hyperthermophile; Protein; Folding; Yeast; Archaea; Expression temperature; Unfolded protein response

Comparison of rat and human alkaline phosphatase isoenzymes and isoforms using HPLC and electrophoresis by Violetta Dziedziejko; Krzysztof Safranow; Dorota Słowik-Żyłka; Anna Machoy-Mokrzyńska; Barbara Millo; Zygmunt Machoy; Dariusz Chlubek (pp. 26-33).

Total activity of alkaline phosphatase (ALP) in serum represents the sum of activities of some isoenzymes and their numerous isoforms derived from various tissues of the organism. The aim of this work was to separate ALP isoenzymes and their isoforms in rat and human serum, compare the properties of serum ALP isoforms in rats and humans, and determine the usefulness of some analytical methods for specific measurements of ALP isoenzyme and isoform activities. Two methods of separation, i.e. high-performance liquid chromatography (HPLC) and agarose gel electrophoresis were chosen. The combination of HPLC with electrophoresis of the eluted fractions and with ALP inhibition methods (urea,l-phenylalanine), inactivation (heat) and precipitation (wheat-germ lectins) enabled the identification of isoenzymes and isoforms of ALP in serum. Using chromatography and a post-column reactor, three isoforms of the intestinal isoenzyme and one bone isoform of a tissue non-specific isoenzyme were detected. Rat serum differs significantly from human as regards activities of intestinal and hepatic isoforms, whereas the properties of the bone isoform are similar in both species. Our HPLC method offers a higher resolution than agarose gel electrophoresis with respect to ALP subfractions in rat serum.

Keywords: Agarose gel electrophoresis; Alkaline phosphatase; High-performance liquid chromatography; Isoenzyme; Isoform; Rats

Identification of serum N-acetylmuramoyl-l-alanine amidase as liver peptidoglycan recognition protein 2 by Yinong Zhang; Leslie van der Fits; Jane S. Voerman; Marie-Jose Melief; Jon D. Laman; Mu Wang; Haitao Wang; Minhui Wang; Xinna Li; Chad D. Walls; Dipika Gupta; Roman Dziarski (pp. 34-46).

N-acetylmuramoyl-l-alanine amidase (NAMLAA) hydrolyzes bacterial peptidoglycan and is present in human serum. A peptidoglycan-recognition protein 2 (PGLYRP2) is expressed in human liver and has N-acetylmuramoyl-l-alanine amidase activity. Here, we determined the amino acid sequences of human serum NAMLAA and liver PGLYRP2 and tested the hypothesis that serum NAMLAA and PGLYRP2 are the same protein. Liver PGLYRP2 and serum NAMLAA had the same mass determined by mass spectrometry and polyacrylamide gel electrophoresis, and both proteins and recombinant PGLYRP2 reacted with polyclonal anti-NAMLAA and anti-PGLYRP2 antibodies, and with monoclonal anti-NAMLAA antibodies. Digestion of serum NAMLAA with trypsin, chymotrypsin, or trypsin plus V8 protease, or with CNBr yielded, respectively, 37, 40, and 3 overlapping peptides that matched 100% and covered 81% of the deduced amino acid sequence of mature PGLYRP2. These peptides overlapped all exon–intron junctions indicating no alternative splice forms. Digestion of liver PGLYRP2 with trypsin yielded 23 peptides that matched 100% and covered 44% of the deduced amino acid sequence of mature PGLYRP2. Serum NAMLAA had a C398–C404 disulfide, partial phosphorylation of S218, and deamidation of N253 and N301. These results indicate that serum NAMLAA and liver PGLYRP2 are the same protein encoded by the pglyrp2 gene.

Keywords: Abbreviations; NAMLAA; N; -acetylmuramoyl-; l; -alanine amidase; NOD; nucleotide-binding oligomerization domain; PGLYRP or PGRP; peptidoglycan-recognition protein; PGN; peptidoglycan; TLR2; Toll-like receptor 2 N; -acetylmuramoyl-; l; -alanine amidase; Bacterial peptidoglycan; Peptidoglycan-recognition protein; Innate immunity

Calorimetric and spectroscopic investigations of the thermal denaturation of wild type nitrite reductase by Andrea Stirpe; Rita Guzzi; Hein Wijma; Martin Ph. Verbeet; Gerard W. Canters; Luigi Sportelli (pp. 47-55).

Nitrite reductase (NiR) is a multicopper protein, with a trimeric structure containing two types of copper site: type 1 is present in each subunit whereas type 2 is localized at the subunits interface. The paper reports on the thermal behaviour of wild type NiR from Alcaligenes faecalis S-6. The temperature-induced changes of the copper centres are characterized by optical spectroscopy and electron paramagnetic resonance spectroscopy, and by establishing the thermal stability by differential scanning calorimetry. The calorimetric profile of the enzyme shows a single endothermic peak with maximum heat absorption at T_m≈100 °C, revealing an exceptional thermal stability. The thermal transition is irreversible and the scan rate dependence of the calorimetric trace indicates that the denaturation of NiR is kinetically controlled. The divergence of the activation energy values determined by different methods is used as a criterion for the inapplicability of the one-step irreversible model. The best fit of the DSC profiles is obtained when the classical Lumry–Eyring model, N⇔U⇒F, is considered. The simulation results indicate that the irreversible step prevails on the reversible one. Moreover, it is found that the conformational changes within the type-1 copper environments precede the denaturation of the whole protein. No evidence of protein dissociation within the temperature range investigated was observed.

Keywords: Nitrite reductase; Thermal denaturation; Activation energy; Lumry–Eyring model

The structure of an enzyme–product complex reveals the critical role of a terminal hydroxide nucleophile in the bacterial phosphotriesterase mechanism by Colin Jackson; Hye-Kyung Kim; Paul D. Carr; Jian-Wei Liu; David L. Ollis (pp. 56-64).

A detailed understanding of the catalytic mechanism of enzymes is an important step toward improving their activity for use in biotechnology. In this paper, crystal soaking experiments and X-ray crystallography were used to analyse the mechanism of the Agrobacterium radiobacter phosphotriesterase, OpdA, an enzyme capable of detoxifying a broad range of organophosphate pesticides. The structures of OpdA complexed with ethylene glycol and the product of dimethoate hydrolysis, dimethyl thiophosphate, provide new details of the catalytic mechanism. These structures suggest that the attacking nucleophile is a terminally bound hydroxide, consistent with the catalytic mechanism of other binuclear metallophosphoesterases. In addition, a crystal structure with the potential substrate trimethyl phosphate bound non-productively demonstrates the importance of the active site cavity in orienting the substrate into an approximation of the transition state.

Keywords: Abbreviations; AChE; acetylcholinesterase; OpdA; organophosphate degrading enzyme; PTE; phosphotriesterase; PEG; polyethylene glycol; EGL; ethylene glycol; DMTP; dimethyl thiophosphate; TMP; trimethyl phosphatePhosphotriesterase; Mechanism; Binuclear; Metalloenzyme; Nucleophile

Single mutation at P1 of a chymotrypsin inhibitor changes it to a trypsin inhibitor: X-ray structural (2.15 �) and biochemical basis by Susmita Khamrui; Jhimli Dasgupta; Jiban K. Dattagupta; Udayaditya Sen (pp. 65-72).

Change in specificity, caused by the mutations at P1 site, of the serine protease inhibitors of different families is reported in the literature, but Kunitz (STI) family inhibitors are almost unexplored in this regard. In this paper, we present the crystal structure of a P1 variant of winged bean chymotrypsin inhibitor (WCI) belonging to Kunitz (STI) family, supplemented by biochemical, phylogenetic and docking studies on the mutant. A single mutation (Leu→Arg) at P1 converted WCI to a strong inhibitor of trypsin with an association constant of 4.8×10¹⁰ M⁻¹ which is comparable to other potent trypsin inhibitors of the family. The crystal structure (2.15 Å) of this mutant (L65R) shows that its reactive site loop conformation deviates from that of WCI and adopts a structure similar to that of Erythrina caffra trypsin inhibitor (ETI) belonging to the same family. Mutation induced structural changes have also been propagated in a concerted manner to the neighboring conserved scaffolding residue Asn14, such that the side chain of this residue took an orientation similar to that of ETI and optimized the hydrogen bonds with the loop residues. While docking studies provide information about the accommodation of non-specific residues in the active site groove of trypsin, the basis of the directional alteration of the reactive site loop conformation has been understood through sequence analysis and related phylogenetic studies.

Keywords: Chymotrypsin inhibitor; Site directed mutagenesis; Specificity change; X-ray structure; Docking study

Probing the substrate specificities of human PHOSPHO1 and PHOSPHO2 by Scott J. Roberts; Alan J. Stewart; Ralf Schmid; Claudia A. Blindauer; Stephanie R. Bond; Peter J. Sadler; Colin Farquharson (pp. 73-82).

PHOSPHO1, a phosphoethanolamine/phosphocholine phosphatase, is upregulated in mineralising cells and is thought to be involved in the generation of inorganic phosphate for bone mineralisation. PHOSPHO2 is a putative phosphatase sharing 42% sequence identity with PHOSPHO1. Both proteins contain three catalytic motifs, conserved within the haloacid dehalogenase superfamily. Mutation of Asp32 and Asp203, key residues within two motifs, abolish PHOSPHO1 activity and confirm it as a member of this superfamily. We also show that Asp43 and Asp123, residues that line the substrate-binding site in our PHOSPHO1 model, are important for substrate hydrolysis. Further comparative modelling reveals that the active sites of PHOSPHO1 and PHOSPHO2 are very similar, but surprisingly, recombinant PHOSPHO2 hydrolyses phosphoethanolamine and phosphocholine relatively poorly. Instead, PHOSPHO2 shows high specific activity toward pyridoxal-5-phosphate ( V_max of 633 nmol min⁻¹ mg⁻¹ and K_m of 45.5 μM). Models of PHOSPHO2 and PHOSPHO1 suggest subtle differences in the charge distributions around the putative substrate entry site and in the location of potential H-bond donors.

Keywords: Abbreviations; CD; circular dichroism; HAD; haloacid dehalogenase; MESG; 2-amino-6-mercapto-7-methylpurine ribonucleoside; Mj; PSP; phosphoserine phosphatase from; Methanococcus jannaschii; Ni-NTA; nickel-nitrilotriacetate; P5P; pyridoxal-5-phosphate; PCho; phosphocholine; PEA; phosphoethanolamine; PNPase; purine nucleoside phophorylase; TBS; Tris-buffered salineBone mineralisation; Haloacid dehalogenase superfamily; Molecular modelling; Phosphatase; Site-directed mutagenesis

Isolation, gene expression and solution structure of a novel moricin analogue, antibacterial peptide from a lepidopteran insect, Spodoptera litura by Yuki Oizumi; Hikaru Hemmi; Masayoshi Minami; Ai Asaoka; Minoru Yamakawa (pp. 83-92).

An antibacterial peptide was isolated from a lepidopteran insect, Spodoptera litura. The molecular mass of this peptide was determined to be 4489.55 by matrix assisted laser desorption/ionization-time of flight mass (MALDI-TOF MS) spectrometry. The peptide consists of 42 amino acids and the sequence has 69–98% identity to those of moricin-related peptides, antibacterial peptides from lepidopetran insects. Thus, the peptide was designated S. litura (Sl) moricin. Sl moricin showed a broad antibacterial spectrum against Gram-positive and negative bacteria. Sl moricin gene was inducible by bacterial injection and expressed tissue—specifically in the fat body and hemocytes. Furthermore, the solution structure of Sl moricin was determined by two-dimensional (2D)¹H-nuclear magnetic resonance (NMR) spectroscopy and hybrid distance geometry-simulated annealing calculation. The tertiary structure revealed a long α-helix containing eight turns along nearly the full length of the peptide like that of moricin, confirming that Sl moricin is a new moricin-like antibacterial peptide. These results suggest that moricin is present not only in B. mori but also in other lepidopteran insects forming a gene family.

Keywords: Antibacterial peptide; Insect immunity; Isolation; Gene expression; NMR; Solution structure; Spodoptera litura

Modifications of protein by polyunsaturated fatty acid ester peroxidation products by Wei Liu; Jin-Ye Wang (pp. 93-98).

The ability of unsaturated fatty acid methyl esters to modify bovine serum albumin (BSA) in the presence of a metal-catalyzed oxidation system [ascorbic acid/Fe (II)/O₂] was investigated. The exposure of BSA to PUFA esters led to the generation of carbonyl groups and the formation of high-molecular-weight proteins, which were strongly dependent on the degree of fatty acid unsaturation. The high-molecular-weight proteins were detected by Western blot analysis and were not recognized by five antibodies. The observation that levels of conjugated dienes and malondialdehyde formed in the presence of BSA were substantially lower in the presence than in the absence of BSA indicated that radicals formed during the degradation of lipid hydroperoxides are likely involved in the formation of protein derivatives. These results may be important in understanding the specific roles of different polyunsaturated fatty acids in the pathophysiological effects associated with oxidative stress.

Keywords: Lipid peroxidation; Protein modification; Western blot analysis

Purification and preliminary crystallographic analysis of a Penicillium expansum lipase by ChuanBing Bian; Cai Yuan; Lin Lin; JunHan Lin; XiaoLi Shi; XiaoMing Ye; ZiXiang Huang; MingDong Huang (pp. 99-102).

PF898 is a strain of Penicillium expansum optimized for the high level production of Penicillium expansum lipase (PEL). This PEL is unique compared with other lipases in several aspects, For example, the PEL shows low sequence identities (<30%) to all other known lipases, and high percentage of hydrophobic residues in the N-terminal region. The PEL was purified to homogeneity and shown to be 28 kDa by SDS-PAGE. Crystals suitable for X-ray diffraction analysis were obtained by the sitting-drop method of vapor diffusion with ammonia sulfate as the precipitating agent at 298 K. The crystals have tetragonal lattice and unit-cell parameters of a=b=88.09 �, c=126.54 �. Diffraction data were collected to a resolution of 2.08 � on an in-house rotating-anode generator.

Keywords: Crystallization; Penicillium expansum; lipase; X-ray crystallography

Overexpression and preliminary X-ray crystallographic analysis of prophenoloxidase activating factor II, a clip domain family of serine proteases by Shunfu Piao; Deukmi Kim; Ji Won Park; Bok Leul Lee; Nam-Chul Ha (pp. 103-106).

A clip domain family of serine proteases has been identified in invertebrates as a crucial enzyme involved in diverse biological processes including immune responses and embryonic development. Although these proteins contain at least one clip domain at the N-terminal of the serine protease domain, the roles and three-dimensional structure of the clip domain are unknown. Prophenoloxidase activating factor-II (PPAF-II), a clip domain family of serine proteases, derived from the beetle Holotrichia diomphalia larvae, was overexpressed in the baculovirus system, and crystallized using the hanging-drop vapor-diffusion method. High-quality single crystals of PPAF-II were obtained in a precipitant solution containing 0.15 M ammonium sulfate, 1.25 M lithium sulfate monohydrate, and 0.1 M sodium citrate dehydrate (pH 5.5). These crystals belong to space group C2 with unit-cell parameters a=107.84, b=76.78, c=70.49 Å and β=113.93°, and contain one or two molecules in the asymmetric unit. Determination of the three-dimensional structure of PPAF-II would clarify the functions of the clip domains.

Keywords: Crystallization; Serine protease; Clip domain; PPAF-II; Masquerade; Overexpression; Baculovirus

Crystallization of AcpA, a respiratory burst-inhibiting acid phosphatase from Francisella tularensis by Richard L. Felts; Thomas J. Reilly; John J. Tanner (pp. 107-110).

Francisella tularensis is a highly infectious bacterial pathogen that is classified as a Category A Pathogen by the Centers for Disease Control and Prevention. Here, we report crystallization of a recombinant form of F. tularensis AcpA, a unique and highly expressed acid phosphatase that is thought to play a role in intracellular survival by inhibiting the host respiratory burst. Three crystal forms have been obtained, with form III being the most suitable for high-resolution structure determination. Form III crystals were grown in the presence of PEG 1500 and the competitive inhibitor sodium orthovanadate (5 mM). The space group is C222(1) with unit cell parameters a=112.1 �, b=144.4 �, c=123.9 �. The asymmetric unit is predicted to contain two protein molecules and 43% solvent. A 1.75-� native data set was recorded at beamline 8.3.1 of the Advanced Light Source. To our knowledge, this is the first report of high-resolution crystals of any F. tularensis protein.

Keywords: Francisella tularensis; Acid phosphatase; AcpA; Biodefense; Crystallization; X-ray diffraction

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BBA - Proteins and Proteomics (v.1752, #1)