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BBA - Proteins and Proteomics (v.1824, #4)

Editorial Board (pp. i).

QM/MM studies on the catalytic mechanism of Phenylethanolamine N-methyltransferase by Q.Q. Hou; J.H. Wang; J. Gao; Y.J. Liu; C.B. Liu (pp. 533-541).

Epinephrine is a naturally occurring adrenomedullary hormone that transduces environmental stressors into cardiovascular actions. As the only route in the catecholamine biosynthetic pathway, Phenylethanolamine N-methyltransferase (PNMT) catalyzes the synthesis of epinephrine. To elucidate the detailed mechanism of enzymatic catalysis of PNMT, combined quantum-mechanical/molecular-mechanical (QM/MM) calculations were performed. The calculation results reveal that this catalysis contains three elementary steps: the deprotonation of protonated norepinphrine, the methyl transferring step and deprotonation of the methylated norepinphrine. The methyl transferring step was proved to be the rate-determining step undergoing a SN2 mechanism with an energy barrier of 16.4kcal/mol. During the whole catalysis, two glutamic acids Glu185 and Glu219 were proved to be loaded with different effects according to the calculations results of the mutants. These calculation results can be used to explain the experimental observations and make a good complementarity for the previous QM study.► We carried out QM/MM calculations to elucidate the reaction mechanism of PNMT. ► The catalysis contains three elementary steps. ► Glu185, Glu219, and a water molecule play an important role in the catalysis.

Keywords: QM/MM; Epinephrine; Methyltransferase; Reaction mechanism; Mutation

Directed evolution of stabilized IgG1-Fc scaffolds by application of strong heat shock to libraries displayed on yeast by Michael W. Traxlmayr; Maximilian Faissner; Gerhard Stadlmayr; Christoph Hasenhindl; Bernhard Antes; Ruker Florian Rüker; Christian Obinger (pp. 542-549).

We have constructed IgG1-Fc scaffolds with increased thermal stability by directed evolution and yeast surface display. As a basis a new selection strategy that allowed the application of yeast surface display for screening of stabilizing mutations in proteins of already high intrinsic thermal stability and T_m-values up to 85°C was developed. Besides library construction by error prone PCR, strong heat stress at 79°C for 10min and screening for well-folded proteins by FACS, sorting rounds had to include an efficient plasmid DNA isolation step for amplification and further transfection. We describe the successful application of this experimental setup for selection of 17 single, double and triple IgG1-Fc variants of increased thermal stability after four selection rounds. The recombinantly produced homodimeric proteins showed a wild-type-like elution profile in size exclusion chromatography as well as content of secondary structures. Moreover, the kinetics of binding of FcRn, CD16a and Protein A to the engineered Fc-molecules was very similar to the wild-type protein. These data clearly demonstrate the importance and efficacy of the presented strategy for selection of stabilizing mutations in proteins of high intrinsic stability within reasonable time.► Construction of IgG1-Fc scaffolds with increased thermal. ► Selection from a yeast displayed protein-library after heat incubation. ► New protocol allowing stabilization of proteins of already high intrinsic stability.

Keywords: Abbreviations; IgG1; immunoglobulin G class 1; Fcab; antigen binding Fc protein; Fc-wt; recombinant wild-type Fc protein; scTCR; single-chain T-cell receptor; MHC; major histocompatibility complex; CDC; complement dependent cytotoxicity; ADCC; antibody dependent cell-mediated cytotoxicity; FcRn; neonatal Fc receptor; CD64; cellular leucocyte Fc receptor FcγRI; DSC; differential scanning calorimetry; ECD; electronic circular dichroism; SEC; size exclusion chromatography; SPR; surface plasmon resonance; RU; response unit; FACS; fluorescent activated cell sorting; PBS; phosphate-buffered salineStability engineering; Antibody engineering; Crystallizable domain; Yeast display; Directed evolution

Proteomic analysis of secretion from human transplanted submandibular gland replacing lacrimal gland with severe keratoconjunctivitis sicca by Bing Song; Lei Zhang; Xiao-jing Liu; Chong Ding; Li-ling Wu; Ye-Hua Gan; Guang-yan Yu (pp. 550-560).

Purpose: Proteomic analysis of secretions from transplanted or non-transplanted submandibular glands in patients with severe keratoconjunctivitis sicca and tears from normal eyes. Experimental design: Secretions from submandibular glands transplanted to replace lacrimal glands and non-transplanted submandibular glands were collected at 1year from 5 patients with severe keratoconjunctivitis sicca undergoing transplantation, and tears were collected from 3 normal subjects. 2-D electrophoresis (2-DE), then mass spectrometry was used to identify proteins. Western blot analysis was used to confirm protein expression. Results: We identified 34 and 11 distinct proteins in the saliva from transplanted submandibular glands and tears, respectively. The saliva from transplanted submandibular glands contained almost all the proteins abundant in tear fluid. The functions of identified proteins in the saliva from transplanted submandibular gland were mainly immune response and anti-bacterial. In total, 7 proteins showed differential expression between the saliva of transplanted and non-transplanted submandibular glands. The upregulation of short palate, lung and nasal epithelium carcinoma-associated protein 2 and carbonic anhydrase VI was confirmed by Western blot analysis. Conclusions: Identified proteins in saliva from transplanted submandibular glands may protect ocular structures. These findings can help in understanding the functional status of transplanted submandibular glands.► We proteomically analysis the saliva from transplanted submandibular gland. ► We found that some proteins differentially expressed after transplantation. ► The upregulation of CA VI and SPLUNC 2 was further confirmed by Western blotting. ► The mechanism of protein synthesis and secretion in transplanted gland has changed.

Keywords: Abbreviations; KCS; Keratoconjunctivitis sicca; 2-D; Two dimensional; SPLUNC2; Short palate, lung and nasal epithelium carcinoma-associated protein 2; CA VI; Carbonic anhydrase VI; CBB; Coomassie brilliant blue; WS; whole saliva; MS; Mass spectrometry; FA; formic acidKeratoconjunctivitis sicca; Proteome; Saliva; Transplantation; Submandibular gland; Tear

Exploring the active site of tripeptidyl-peptidase II through studies of pH dependence of reaction kinetics by Sandra Eklund; Lindas Ann-Christin Lindås; Emil Hamnevik; Mikael Widersten; Birgitta Tomkinson (pp. 561-570).

Tripeptidyl-peptidase II (TPP II) is a subtilisin-like serine protease which forms a large enzyme complex (>4MDa). It is considered a potential drug target due to its involvement in specific physiological processes. However, information is scarce concerning the kinetic characteristics of TPP II and its active site features, which are important for design of efficient inhibitors. To amend this, we probed the active site by determining the pH dependence of TPP II catalysis. Access to pure enzyme is a prerequisite for kinetic investigations and herein we introduce the first efficient purification system for heterologously expressed mammalian TPP II. The pH dependence of kinetic parameters for hydrolysis of two different chromogenic substrates, Ala-Ala-Phe- pNA and Ala-Ala-Ala- pNA, was determined for murine, human and Drosophila melanogaster TPP II as well as mutant variants thereof. The investigation demonstrated that TPP II, in contrast to subtilisin, has a bell-shaped pH dependence of k_cat^app/K_M probably due to deprotonation of the N-terminal amino group of the substrate at higher pH. Since both the K_M and k_cat^app are lower for cleavage of AAA- pNA than for AAF- pNA we propose that the former can bind non-productively to the active site of the enzyme, a phenomenon previously observed with some substrates for subtilisin. Two mutant variants, H267A and D387G, showed bell-shaped pH-dependence of k_cat^app, possibly due to an impaired protonation of the leaving group. This work reveals previously unknown differences between TPP II orthologues and subtilisin as well as features that might be conserved within the entire family of subtilisin-like serine peptidases.► The first system for purifying mammalian TPP II expressed in E. coli is presented. ► Through determination of pH-dependence the active site of TPP II is investigated. ► The pH-dependence of k_cat/K_M is affected by substrate protonation state. ► Evidence for non-productive binding of some substrates in TPP II is revealed. ► The microenvironment in the active site is more charged in insect TPP II.

Keywords: Abbreviations; AAA-; p; NA; alanyl-alanyl-alanyl-; para; -nitroanilide; AAF-; p; NA; alanyl-alanyl-phenylalanyl-; para-; nitroanilide; HIC; hydrophobic interaction chromatography; IPTG; isopropyl-β-D-1-thiogalactopyranoside; PEI; polyethyleneimine; suc-AAPF-; p; NA; succinyl-alanyl-alanyl-prolyl-phenylalanyl-; para; -nitroanilide; TPP II; tripeptidyl-peptidase II (the species is indicated by a lower case letter: m (murine), h (human) or d (; Drosophila melanogaster; ))Tripeptidyl-peptidase II; AAF-; p; NA; AAA-; p; NA; Steady-state kinetics; pH-dependence

Exploring the unfolding mechanism of γ-glutamyltranspeptidases: The case of the thermophilic enzyme from Geobacillus thermodenitrificans by Andrea Pica; Irene Russo Krauss; Immacolata Castellano; Mosè Rossi; Francesco La Cara; Giuseppe Graziano; Filomena Sica; Antonello Merlino (pp. 571-577).

γ-glutamyltranspeptidases (γ-GTs) are ubiquitous enzymes that catalyze the hydrolysis of γ-glutamyl bonds in glutathione and glutamine and the transfer of the released γ-glutamyl group to amino acids or short peptides. These enzymes are generally synthesized as precursor proteins, which undergo an intra-molecular autocatalytic cleavage yielding a large and a small subunit. In this study, circular dichroism and intrinsic fluorescence measurements have been used to investigate the structural features and the temperature- and guanidinium hydrochloride (GdnHCl)-induced unfolding of the mature form of the γ-GT from Geobacillus thermodenitrificans ( GthGT) and that of its T353A mutant , which represents a mimic of the precursor protein . Data indicate that a) the mutant and the mature GthGT have a different secondary structure content and a slightly different exposure of hydrophobic regions, b) the thermal unfolding processes of both GthGT forms occur through a three-state model, characterized by a stable intermediate species, whereas chemical denaturations proceed through a single transition, c) both GthGT forms exhibit remarkable stability against temperature, but they do not display a strong resistance to the denaturing action of GdnHCl. These findings suggest that electrostatic interactions significantly contribute to the protein stability and that both the precursor and the mature form of GthGT assume compact and stable conformations to resist to the extreme temperatures where G. thermodenidrificans lives. Owing to its thermostability and unique catalytic properties, GthGT is an excellent candidate to be used as a glutaminase in food industry.Display Omitted► The thermal and chemical stability of GthGT and its T353A mutant has been reported. ► An intermediate has been detected during the thermal unfolding of GthGT and T353A. ► Mature GthGT is more susceptible at acidic pH to chemical denaturation than T353A. ► Electrostatics are important for the stability of GthGT.

Keywords: Abbreviations; ANS; 1-anilino-8-naphthalenesulfonate; Bl; GT; γ-glutamyltranspeptidase from; Bacillus licheniformis; Bs; GT; γ-glutamyltranspeptidase from; Bacillus subtilis; CD; circular dichroism; GdnHCl; guanidinium hydrochloride; Ec; GT; γ-glutamyltranspeptidase from; Escherichia coli; GGT; γ-glutamyltranspeptidase or (γ-glutamyl)-peptide: amino acid γ-glutamyltransferase (EC 2.3.2.2); Gth; GT; γ-glutamyltranspeptidase from; Geobacillus thermodenitrificans; GSH; reduced glutathione; Hp; GT; γ-glutamyltranspeptidase from; Helicobacter pylori; , EDTA, ethylene-diamine-tetra-acetic acidThermal unfolding; Intermediate state; Chemical denaturation; Circular dichroism; γ-glutamyltranspeptidases; Protein stability

The dehaloperoxidase paradox by Stefan Franzen; Matthew K. Thompson; Reza A. Ghiladi (pp. 578-588).

The dual functions of the dehaloperoxidase-hemoglobin of Amphitrite ornata leads to a paradox. Peroxidase and hemoglobin functions require ferric and ferrous resting states, respectively. Assuming that hemoglobin function is the dominant function, the starting point for peroxidase activation would be the oxyferrous state. Activation of that state leads to the ferryl intermediate, followed by one-electron oxidation of the substrate, which results in the ferric state. Since no exogenous reductant is known, there is no return to the ferrous form or hemoglobin function. The observation that an internal binding site for 4-bromophenol leads to inhibition leads to a further paradox that the enzyme would be inhibited immediately upon activation under ambient conditions in benthic ecosystems where the inhibitor, 4-bromophenol is present in greater concentration than the substrate, 2,4,6-tribromophenol. In this review, we explore the unresolved aspects of the reaction scheme that leads to the apparent paradox. Recent data showing activation of the oxyferrous state, an extremely high reduction potential and exogenous reduction by the 2,6-dibromoquinone product present a potential resolution of the paradox. These aspects are discussed in the context of control of reactivity radical pathways and reactivity by the motion of the distal histidine, H55, which in turn is coupled to the binding of substrate and inhibitor.► Dual functions of protein have mutually exclusive ferrous and ferric iron states. ► Internal binding of an inhibitor competes with external substrate binding. ► Radical pathways under control of protein conformation. ► Distal histidine flexibility responsible for multiple functions. ► Structural confirmation by X-ray crystal structures with inhibitors and Xe.

Keywords: Peroxidase; Competitive inhibition; Hemoglobin; Phenol; Marine; Enzyme

Mutational analysis of cysteine 328 and cysteine 368 at the interface of Plasmodium falciparum adenylosuccinate synthetase by Sonali Mehrotra; Mylarappa B.Ningappa; Jayalakshmi Raman; Ranjith P. Anand; Hemalatha Balaram (pp. 589-597).

Plasmodium falciparum adenylosuccinate synthetase, a homodimeric enzyme, contains 10 cysteine residues per subunit. Among these, Cys250, Cys328 and Cys368 lie at the dimer interface and are not conserved across organisms. PfAdSS has a positively charged interface with the crystal structure showing additional electron density around Cys328 and Cys368. Biochemical characterization of site directed mutants followed by equilibrium unfolding studies permits elucidation of the role of interface cysteines and positively charged interface in dimer stability. Mutation of interface cysteines, Cys328 and Cys368 to serine, perturbed the monomer–dimer equilibrium in the protein with a small population of monomer being evident in the double mutant. Introduction of negative charge in the form of C328D mutation resulted in stabilization of protein dimer as evident by size exclusion chromatography at high ionic strength buffer and equilibrium unfolding in the presence of urea. These observations suggest that cysteines at the dimer interface of PfAdSS may indeed be charged and exist as thiolate anion. ► P. falciparum (Pf) adenylosuccinate synthetase (AdSS) is a homodimeric enzyme. ► Dimer interface of PfAdSS is distinct from E. coli and mouse AdSS. ► Enzyme has a preponderance of positively charged residues at dimer interface. ► Crystal structure shows additional electron density around interface cysteines. ► Stability studies on interface cysteine mutants suggest role for thiolate anion.

Keywords: Abbreviations; AdSS; Adenylosuccinate synthetase; PfAdSS; P. falciparum; adenylosuccinate synthetase; WtAdSS; wildtype adenylosuccinate synthetase; IMP; inosine monophosphate; GTP; guanosine triphosphate; IAA; iodoacetate P. falciparum; Adenylosuccinate synthetase; Interface cysteines; Site-directed mutagenesis; Urea induced protein denaturation; Folding intermediate

Extracellular tyrosinase from the fungus Trichoderma reesei shows product inhibition and different inhibition mechanism from the intracellular tyrosinase from Agaricus bisporus by Chiara Gasparetti; Emilia Nordlund; Janis Janne Jänis; Johanna Buchert; Kristiina Kruus (pp. 598-607).

Tyrosinase (EC 1.14.18.1) is a widely distributed type 3 copper enzyme participating in essential biological functions. Tyrosinases are potential biotools as biosensors or protein crosslinkers. Understanding the reaction mechanism of tyrosinases is fundamental for developing tyrosinase-based applications. The reaction mechanisms of tyrosinases from Trichoderma reesei (TrT) and Agaricus bisporus (AbT) were analyzed using three diphenolic substrates: caffeic acid, L-DOPA (3,4-dihydroxy-l-phenylalanine), and catechol. With caffeic acid the oxidation rates of TrT and AbT were comparable; whereas with L-DOPA or catechol a fast decrease in the oxidation rates was observed in the TrT-catalyzed reactions only, suggesting end product inhibition of TrT. Dopachrome was the only reaction end product formed by TrT- or AbT-catalyzed oxidation of L-DOPA. We produced dopachrome by AbT-catalyzed oxidation of L-DOPA and analyzed the TrT end product (i.e. dopachrome) inhibition by oxygen consumption measurement. In the presence of 1.5mM dopachrome the oxygen consumption rate of TrT on 8mM L-DOPA was halved. The type of inhibition of potential inhibitors for TrT was studied using p-coumaric acid (monophenol) and caffeic acid (diphenol) as substrates. The strongest inhibitors were potassium cyanide for the TrT-monophenolase activity, and kojic acid for the TrT-diphenolase activity. The lag period related to the TrT-catalyzed oxidation of monophenol was prolonged by kojic acid, sodium azide and arbutin; contrary it was reduced by potassium cyanide. Furthermore, sodium azide slowed down the initial oxidation rate of TrT- and AbT-catalyzed oxidation of L-DOPA or catechol, but it also formed adducts with the reaction end products, i.e., dopachrome and o-benzoquinone.► Trichoderma reesei tyrosinase suffers from dopachrome product inhibition. ► Sodium azide forms azido-quinone adducts with the tyrosinase reaction end products. ► Potassium cyanide and kojic acid are strong inhibitors of T. reesei tyrosinase.

Keywords: Tyrosinase; Trichoderma reesei; Agaricus bisporus; End product inhibition; Dopachrome; K; _i

Identification of the critical structural determinants of the EF-hand domain arrangements in calcium binding proteins by Ye-dan Feng; Jing Li; Wen-chang Zhou; Zhi-guang Jia; Qun Wei (pp. 608-619).

EF-hand calcium binding proteins (CaBPs) share strong sequence homology, but exhibit great diversity in structure and function. Thus although calmodulin (CaM) and calcineurin B (CNB) both consist of four EF hands, their domain arrangements are quite distinct. CaM and the CaM-like proteins are characterized by an extended architecture, whereas CNB and the CNB-like proteins have a more compact form. In this study, we performed structural alignments and molecular dynamics (MD) simulations on 3 CaM-like proteins and 6 CNB-like proteins, and quantified their distinct structural and dynamical features in an effort to establish how their sequences specify their structures and dynamics. Alignments of the EF2–EF3 region of these proteins revealed that several residues (not restricted to the linker between the EF2 and EF3 motifs) differed between the two groups of proteins. A customized inverse folding approach followed by structural assessments and MD simulations established the critical role of these residues in determining the structure of the proteins. Identification of the critical determinants of the two different EF-hand domain arrangements and the distinct dynamical features relevant to their respective functions provides insight into the relationships between sequence, structure, dynamics and function among these EF-hand CaBPs.► We compare two groups of EF-hand proteins mainly by molecular dynamics simulations. ► Distinct features on structures and dynamics of two groups are quantified. ► Critical residues in the EF2–EF3 region are found determining the architecture. ► A customized inverse folding approach verifies the importance of these residues.

Keywords: Abbreviations; Ca; ²⁺; calcium; CaBPs; calcium binding proteins; CaM; calmodulin; CNB; calcineurin B; SCP; sarcoplasmic calcium binding protein; TnC; troponin C; TnI; troponin I; TnT; troponin T; Tn; troponin; hCLP; human calmodulin-like protein; CIB1; calcium and integrin binding protein 1; CHPs; calcineurin B homologous proteins; Frq1; yeast frequenin; NCS; neuronal calcium sensor; GCAPs; guanylyl cyclase activating proteins; KChIPs; Kv channel interacting proteins; RECO; recoverin; CNA; calcineurin A; NHE1; Na; ⁺; /H; ⁺; exchanger 1; Pik1; phosphatidylinositol 4 kinase; PDB; protein data bank; MD; molecular dynamics; RMSD; root mean square deviation; PME; particle mesh Ewald; ED; essential dynamics; PCA; principal component analysisEF-hand calcium binding protein; Calmodulin; Calcineurin B; Structural determinant; Molecular dynamics

Gly or Ala substitutions for Pro²¹⁰Thr²¹¹Asn²¹² at the β8–β9 turn of subtilisin Carlsberg increase the catalytic rate and decrease thermostability by Naoki Fuchita; Saori Arita; Junya Ikuta; Masahiro Miura; Kaori Shimomura; Hiroyuki Motoshima; Keiichi Watanabe (pp. 620-626).

A comparison of the primary structures among psychrophilic, mesophilic, and thermophilic subtilases revealed that the turn between the β8 and β9 strands (β8–β9 turn, BPN′ numbering) of psychrophilic subtilases are more flexible than those of their mesophilic and thermophilic counterparts. To investigate the relationship between structure of this turn and enzyme activity as well as thermostability of mesophilic subtilisin Carlsberg (sC), we analyzed 6 mutants of sC with a single, double, or triple Gly or Ala substitutions for Pro²¹⁰Thr²¹¹Asn²¹² at the β8–β9 turn. Among the single Gly substitutions, the P210G substitution most significantly (1.5-fold) increased the specific activity on N-succinyl-Ala-Ala-Pro-Phe- p-nitroanilide (AAPF) substrate and 12-fold decreased the thermostability. All mutants tested showed the increased k_cat for the AAPF substrate and reduced thermostability compared with the wild-type sC. The k_cat values of the P210G, P210G/T211G, and P210G/T211G/N212G mutants were 1.5-, 1.7-, and 1.8-fold higher than that of the wild-type sC. There were significant positive correlations between k_cat and thermal inactivation rates as well as k_cat and K_m of the wild-type and mutants. These results demonstrate that the structure of β8–β9 turn, despite its distance from the active site, has significant effects on the catalytic rate and thermostability of sC through a global network of intramolecular interactions and suggest that the lack of flexibility of this turn stabilizes the wild-type sC against thermal inactivation in compensation for some loss of catalytic activity.► We substituted Gly or Ala for Pro-Thr-Asn at β8–β9 turn of subtilisin Carlsberg (sC). ► This turn is located apart from the active site. ► These mutations increased k_cat for peptide hydrolysis and decreased thermostability. ► P210G mutation had the largest effects among single Gly substitutions for PTN residues. ► Positive correlations were found between k_cat and thermal inactivation rates.

Keywords: Abbreviations; sC; subtilisin Carlsberg; AAPF; N; -succinyl-Ala-Ala-Pro-Phe-; p; -nitroanilide; AAPL; N; -succinyl-Ala-Ala-Pro-Leu-; p; -nitroanilide; AAVA; N; -succinyl-Ala-Ala-Val-Ala-; p; -nitroanilide; PTN; wild-type; GTN; P210G mutant; PGN; T211G mutant; PTG; N212G mutant; ATN; P210A mutant; GGN; P210G/T211G mutant; GGG; P210G/T211G/N212G mutantSubtilisin; Catalytic rate; Thermostability; β turn; Proline; Flexibility

A rational approach to the regioselective deacetylation of 2′,3′,5′-tri- O-acetyluridine by Novozym 435 catalysed alcoholysis by Gudino E.D. Gudiño; L.E. Iglesias; M.L. Ferreira (pp. 627-636).

To give a rational explanation for the behaviour of 2′,3′,5′-tri- O-acetyluridine (TAU) catalysed alcoholysis using Novozym 435, the commercial biocatalyst with immobilized Candida antarctica lipase B (CALB), a set of experiments analyzing the role of the alcohol/substrate (A/S) molar ratio, alcohol/biocatalyst (A/B) and substrate/biocatalyst (S/B) mass ratios were carried out. At a A/S=120 and a S/B=6.16, 2′,3′-di- O-acetyluridine (DAU) was obtained in 92% at 22h. The observed trend towards the exclusive formation of DAU at very high alcohol amounts can be explained on the basis of the change of substrate orientation from normal to inverse. The simple molecular modelling analysis supports that key O/H atoms from TAU and the resulting intermediates display the adequate distances to generate productive binding only when the inverse coordination of TAU is present through the 5′-moiety of TAU, at high ethanol concentrations. At these conditions a possible allosteric-like effect of ethanol, combined with water in an H-network in the catalytic triad and in its neighbourhood, could explain the high selectivity towards the production of DAU at selected conditions.► Molecular modeling tools applied to acetylated nucleosides ethanolysis with Novozym 435. ► MM2 study of the interaction of substrates from acetylated nucleosides in reaction with ethanol for CALB. ► Correlation experimental results with Novozym 435 with theoretical approach on CALB. ► Analysis and discussion of role of ethanol in regioselectivity of CALB (as Novozym 435).

Keywords: Candida antarctica; lipase B; Molecular modeling; Nucleoside; Regioselectivity; Transesterification

Charged single alpha-helices in proteomes revealed by a consensus prediction approach by Gaspari Zoltán Gáspári; Suveges Dániel Süveges; András Perczel; László Nyitray; Toth Gábor Tóth (pp. 637-646).

Charged single α-helices (CSAHs) constitute a recently recognized protein structural motif. Its presence and role is characterized in only a few proteins. To explore its general features, a comprehensive study is necessary. We have set up a consensus prediction method available as a web service (athttp://csahserver.chem.elte.hu) and downloadable scripts capable of predicting CSAHs from protein sequences. Using our method, we have performed a comprehensive search on the UniProt database. We found that the motif is very rare but seems abundant in proteins involved in symbiosis and RNA binding/processing. Although there are related proteins with CSAH segments, the motif shows no deep conservation in protein families. We conclude that CSAH-containing proteins, although rare, are involved in many key biological processes. Their conservation pattern and prevalence in symbiosis-associated proteins suggest that they might be subjects of relatively rapid molecular evolution and thus can contribute to the emergence of novel functions.► The charged single α-helix (CSAH) is a novel protein structural motif. ► Using a consensus prediction approach, we surveyed all UniProt proteins. ► The CSAH motif is most abundant in RNA-binding and symbiosis-related proteins. ► The motif shows no deep conservation in protein families. ► CSAH segments might be subjects of relatively rapid molecular evolution.

Keywords: Charged single alpha-helix; Structure prediction; Protein evolution

Kinetic characterisation of o-aminophenols and aromatic o-diamines as suicide substrates of tyrosinase by Munoz-Munoz Jose Luis Muñoz-Muñoz; Francisco Garcia-Molina; Jose Berna; Pedro Antonio Garcia-Ruiz; Ramon Varon; Jose Tudela; Jose N. Rodriguez-Lopez; Francisco Garcia-Canovas (pp. 647-655).

We study the suicide inactivation of tyrosinase acting on o-aminophenols and aromatic o-diamines and compare the results with those obtained for the corresponding o-diphenols. The catalytic constants follow the order aromatic o-diamines< o-aminophenols< o-diphenols, which agrees with the view that the transfer of the proton to the peroxide group of the oxy-tyrosinase form is the slowest step in the catalytic cycle. As regards the apparent inactivation constant, it remains within the same order of magnitude, although slightly lower in the case of the aromatic o-diamines and o-aminophenols than o-diphenols: o-diamines< o-aminophenols< o-diphenols. The efficiency of the second nucleophilic attack of substrate on CuA seems to be the determining factor in the bifurcation of the inactivation and catalytic pathways. This attack is more efficient in o-diamines (where it attacks a nitrogen atom) than in o-aminophenols and o-diphenols (where it attacks an oxygen atom), favouring the catalytic pathway and slowing down the inactivation pathway. The inactivation step is the slowest of the whole process. The values of r, the number of turnovers that 1mol of enzyme carries out before being inactivated, follows the order aromatic o-diamines< o-aminophenols< o-diphenols. As regards the Michaelis constants, that of the o-diamines is slightly lower than that of the o-diphenols, while that of the o-aminophenols is slightly greater than that observed for the o-diphenols. As a consequence of the above, the inactivation efficiency, λ_max/ K_m^S, follows this order: o-diphenols> o-aminophenols>aromatic o-diamines.► This study describes the importance of proton transfer from the substrates to oxy-tyrosinase. ► During the catalysis, one proton is transferred to base B₂ and other to the peroxide group. ► The transfer of two protons to the peroxide group probably occurs during suicide inactivation. ► The limiting step of the catalysis could be the transfer of the first proton to the peroxide group. ► The limiting step for inactivation could be second proton transfer to the peroxide group.

Keywords: Tyrosinase; Aromatic; o-; diamine; o-; Aminophenol; Suicide inactivation; o-; Diphenol

Toxicological biomarkers of 2,3,4,7,8-pentachlorodibenzofuran in proteins secreted by HepG2 cells by Sohee Phark; So-Young Park; Seonyoung Choi; Zhi Zheng; Eunkyung Cho; Min Lee; Ji-youn Lim; Jong Bok Seo; Nam Hee Won; Woon-Won Jung; Donggeun Sul (pp. 656-666).

Using a proteomic approach, a study was conducted for determination of the effects of 2,3,4,7,8-pentachlorodibenzofuran (2,3,4,7,8-PCDF) on proteins secreted by HepG2 cells. Briefly, HepG2 cells were exposed to various concentrations of 2,3,4,7,8-PCDF for 24 or 48h. MTT and comet assays were then conducted for determination of cytotoxicity and genotoxicity, respectively. Results of an MTT assay showed that 1nM of 2,3,4,7,8-PCDF was the maximum concentration that did not cause cell death. In addition, a dose- and time dependent increase of DNA damage was observed in HepG2 cells exposed to 2,3,4,7,8-PCDF. Therefore, two different concentrations of 2,3,4,7,8-PCDF, 1 and 5nM, were selected for further analysis of proteomic biomarkers using two different pI ranges (4–7 and 6–9) and large two dimensional gel electrophoresis. Results showed identification of 32 proteins ( 29 up- and 3 down-regulated) by nano-LC-ESI-MS/MS and nano-ESI on a Q-TOF2 MS. Among these, the identities of pyridoxine-5'-phosphate oxidase, UDP-glucose 6-dehydrogenase, plasminogen activator inhibitor I precursor, plasminogen activator inhibitor-3, proteasome activator complex subunit 1, isoform 1 of 14-3-3 protein sigma, peptidyl-prolyl cis-trans isomerase A, 14-3-3 protein gamma, protein DJ-1, and nucleoside diphosphate kinase A were confirmed by western blot analysis. The differential expression of protein DJ-1, proteasome activator complex subunit 1 and plasminogen activator inhibitor-3 was further validated in plasma proteins from rats exposed to 2,3,4,7,8-PCDF. These proteins could be used as potential toxicological biomarkers of 2,3,4,7,8-PCDF.► 2,3,4,7,8-PCDF is the most abundant molecule in dioxin-like compounds. ► The toxicological biomarkers of 2,3,7,8-TCDD in secreted proteins were evaluated using HepG2 cells. ► Protein DJ-1, proteasome activator complex subunit 1 and plasminogen activator inhibitor-3 were validated in rat plasma. ► These proteins could be used as potential toxicological biomarkers of 2,3,4,7,8-PCDF.

Keywords: Abbreviations; 2,3,4,7,8-PCDF; 2,3,4,7,8-pentachlorodibenzofuran; MTT; 3-(4,5-Dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide; LC-ESI-MS/MS; liquid chromatography electrospray ionization-tandem mass spectrometry; TOF2 MS; time-of-flight 2 mass spectrometry; PCDFs; polychlorinated dibenzofurans; NMA; normal point melting agarose; LMA; low point melting agarose; IEF; isoelectric focusing; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; 2-DE; two dimensional gel electrophoresis; PNPO; pyridoxine-5′-phosphate oxidase; UDP-GlcDH; UDP-glucose 6-dehydrogenase; PAI-1; plasminogen activator inhibitor I precursor; PAI-3; plasminogen activator inhibitor-3 precursor; PA28α; proteasome activator complex subunit 1; 14-3-3 σ; isoform 1 of 14-3-3 protein sigma; PPIase A; peptidyl-prolyl cis-trans isomerase A; 14-3-3 υ; 14-3-3 protein gamma; DJ-1; protein DJ-1; NDK A; nucleoside diphosphate kinase A2,3,4,7,8-pentachlorodibenzofuran; Biomarker; HepG2 cell; Proteomic; Rat plasma; Two-dimensional gel electrophoresis

HIV-1 p6—Another viral interaction partner to the host cellular protein cyclophilin A by Sara M.Ø. Solbak; Tove R. Reksten; Roder Rene Röder; Victor Wray; Ole Horvli; Arnt J. Raae; Petra Henklein; Peter Henklein; Torgils Fossen (pp. 667-678).

The 52-amino acid human immunodeficiency virus type 1 (HIV-1) p6 protein has previously been recognized as a docking site for several cellular and viral binding factors and is important for the formation of infectious viruses. A particular structural feature of p6 is the notably high relative content of proline residues, located at positions 5, 7, 10, 11, 24, 30, 37 and 49 in the sequence. Proline cis/trans isomerism was detected for all these proline residues to such an extent that more than 40% of all p6 molecules contain at least one proline in a cis conformation. 2D¹H nuclear magnetic resonance analysis of full-length HIV-1 p6 and p6 peptides established that cyclophilin A (CypA) interacts as a peptidyl–prolyl cis/trans isomerase with all proline residues of p6. Only catalytic amounts of CypA were necessary for the interaction with p6 to occur, strongly suggesting that the observed interaction is highly relevant in vivo. In addition, surface plasmon resonance studies revealed binding of full-length p6 to CypA, and that this binding was significantly stronger than any of its N- or C-terminal peptides. This study demonstrates the first identification of an interaction between HIV-1 p6 and the host cellular protein CypA. The mode of interaction involves both transient enzyme–substrate interactions and a more stable binding. The binding motifs of p6 to Tsg-101, ALIX and Vpr coincide with binding regions and catalytic sites of p6 to CypA, suggesting a potential role of CypA in modulating functional interactions of HIV-1.► HIV-1 p6 has been identified to interact with human cyclophilin A. ► Cyclophilin A generally catalyzes all Pro isomerizations of a full-length protein. ► CypA may catalyse any Pro isomerization in any protein in solution in vivo. ► Thereby a multidimensional catalytic universe is introduced.

Keywords: Abbreviations; ALIX; ALG-2 interacting protein 1/X; CA; capsid; CsA; cyclosporine A; CypA; cyclophilin A; DMSO; dimethyl sulfoxide; HIV-1; human immunodeficiency virus type 1; L-domains; late assembly domains; NMR; nuclear magnetic resonance; NOESY; Nuclear Overhauser Effect Spectroscopy; PPIases; peptidyl–prolyl cis/trans-isomerases; Pro; proline; ROESY; Rotating-Frame Overhauser Effect Spectroscopy; SPR; surface plasmon resonance; TOCSY; Total Correlation Spectroscopy; Tsg101; tumor susceptibility gene 101; VLP; Virus like particles; Vpr; viral protein RHIV-1 p6; Cyclophilin A; NMR; Biacore; SPR; Peptidyl–prolyl isomerase

Crystal structures of a type-1 ribosome inactivating protein from Momordica balsamina in the bound and unbound states by Gajraj Singh Kushwaha; Nisha Pandey; Mau Sinha; S. Baskar Singh; Punit Kaur; Sujata Sharma; Tej P. Singh (pp. 679-691).

The ribosome inactivating proteins (RIPs) of type 1 are plant toxins that eliminate adenine base selectively from the single stranded loop of rRNA. We report six crystal structures, type 1 RIP from Momordica balsamina (A), three in complexed states with ribose (B), guanine (C) and adenine (D) and two structures of MbRIP-1 when crystallized with adenosine triphosphate (ATP) (E) and 2′-deoxyadenosine triphosphate (2′-dATP) (F). These were determined at 1.67Å, 1.60Å, 2.20Å, 1.70Å, 2.07Å and 1.90Å resolutions respectively. The structures contained, (A) unbound protein molecule, (B) one protein molecule and one ribose sugar, (C) one protein molecule and one guanine base, (D) one protein molecule and one adenine base, (E) one protein molecule and one ATP-product adenine molecule and (F) one protein molecule and one 2′-dATP-product adenine molecule. Three distinct conformations of the side chain of Tyr70 were observed with (i) χ¹=−66°and χ²=165° in structures (A) and (B); (ii) χ¹=−95° and χ²=70° in structures (C), (D) and (E); and (iii) χ¹=−163° and χ²=87° in structure (F). The conformation of Tyr70 in (F) corresponds to the structure of a conformational intermediate. This is the first structure which demonstrates that the slow conversion of DNA substrates by RIPs can be trapped during crystallization.► The isolated new type 1 ribosome inactivating protein MbRIP-1 is highly toxic. ► Ribose, guanine, ATP and 2′dATP bind to MbRIP-1 at substrate binding site. ► The conformation of active site is unaltered in the complex with ribose. ► The conformation of active site is altered in complexes with guanine and adenine. ► The conformation of active site is further altered when complexed with 2′dATP product.

Keywords: Ribosome inactivating protein; Crystal structure; Complex; Ribose; Guanine; Adenine intermediate

Differential proteome profiling of pleural effusions from lung cancer and benign inflammatory disease patients by Zhengyang Wang; Cheng Wang; Xiaobin Huang; Ying Shen; Jing Shen; Kejing Ying (pp. 692-700).

The pleural effusion proteome has been found containing information that directly reflects pathophysiological status and represents a potential diagnostic value for pulmonary diseases. However, the variability in protein composition between malignant and benign effusions is not well understood. Herein, we investigated the changes of proteins in pleural effusions from lung adenocarcinoma and benign inflammatory disease (pneumonia and tuberculosis) patients by two-dimensional difference gel electrophoresis (2D-DIGE). Twenty-eight protein spots displayed significantly different expression levels were positively identified by MALDI-TOF-MS representing 16 unique proteins. Five identified protein candidates were further validated and analyzed in effusions, sera or tissues. Among them, hemopexin, fibrinogen gamma and transthyretin (TTR) were up-regulated in cancer samples. The effusion concentration of serum amyloid P component (SAP) was significantly lower in lung cancer patients than in benign inflammatory patients, but no differences were found in sera samples. Moreover, a Jumonji C (JmjC)-domain-containing protein, JMJD5, was observed to be down-regulated in malignant effusions, lung cancer tissues and cancer cells. These results shed light on the altered pleural effusion proteins as a useful and important complement to plasma or other routine clinical tests for pulmonary disease diagnosis.Display Omitted► Differential 2-D DIGE analysis of malignant and benign inflammatory pleural effusions. ► Sixteen differentially expressed proteins were identified by mass spectrometry. ► Five proteins were validated and analyzed in effusions, sera, tissues or cancer cells. ► Hemopexin, fibrinogen gamma and TTR were up-regulated in cancer samples. ► SAP and JMJD5 were down-regulated in cancer samples.

Keywords: Pleural effusion; Lung cancer; Benign inflammatory lung disease; 2D-DIGE

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BBA - Proteins and Proteomics (v.1824, #4)