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BBA - Proteins and Proteomics (v.1834, #1)

Editorial Board (pp. i).

Reviewer Acknowledgment (pp. iii-v).

Characterization of the oligomerization and ligand-binding properties of recombinant rat lipocalin 11 by Yina Gu; Qiang Liu; Peiyan Chen; Chenyun Guo; Yan Liu; Yufen Zhao; Yonglian Zhang; Donghai Lin (pp. 1-7).

Lipocalin 11 (Lcn11), a recently identified member of the lipocalin family, potentially plays crucial physiological roles in male reproduction. In this present work, we cloned, expressed and purified the rat Lcn11 (rLcn11) protein in Escherichia coli. A C59A/C156A substitution was introduced to ameliorate the misfolding and aggregation problem associated with the wild-type protein. From circular dichroism and non-reducing SDS–PAGE, we characterized the conformational properties of rLcn11 as a typical lipocalin scaffold with the conserved disulfide bridge. The results obtained from size-exclusion chromatography, cross-linking experiment and dynamic light scattering analysis indicate that the recombinant rLcn11 protein forms dimer in neutral solution. By using fluorescent probe 8-anilino-1-naphtahlene sulfonic acid (ANS), we found rLcn11 might contain multiple hydrophobic binding sites for ligand binding. Similarly to the odorant-binding protein, rLcn11 processes a moderate affinity for binding 1-aminoanthracene (AMA), implying that Lcn11 might work as a dimeric chemoreception protein in male reproductive system.► An efficient approach to express and purify rLcn11 protein and C59A/C156A mutant. ► The first study of the physicochemical properties of rLcn11 protein. ► The recombinant rLcn11 protein forms dimer in neutral solution. ► The C59A/C156A rLcn11 processes multiple binding sites for hydrophobic ligand. ► A moderate binding affinity of C59A/C156A rLcn11 for binding ligand AMA.

Keywords: Abbreviations; rLcn11; rat lipocalin 11; L-PGDS; lipocalin-type prostaglandin D synthase; OBP; odorant-binding protein; MUP; major urine protein; PBP; pheromone-binding proteins; WT; wild-type; LB; Luria-Bertani; IPTG; isopropyl β-; d; -thiogalactoside; MALDI-TOF; matrix-assisted laser desorption ionization time-of-flight; DLS; dynamic light scattering; EGS; Ethylene glycolbis (succinimidylsuccinate); CD; circular dichroismLipocalin; Lcn11; Dimer; Disulfide bond; Ligand-binding; β-barrel

Proteomics in reproductive biology: Beacon for unraveling the molecular complexities by Rahul D. Upadhyay; N.H. Balasinor; Anita V. Kumar; Geetanjali Sachdeva; Priyanka Parte; Kushaan Dumasia (pp. 8-15).

Proteomics, an interface of rapidly evolving advances in physics and biology, is rapidly developing and expanding its potential applications to molecular and cellular biology. Application of proteomics tools has contributed towards identification of relevant protein biomarkers that can potentially change the strategies for early diagnosis and treatment of several diseases. The emergence of powerful mass spectrometry-based proteomics technique has added a new dimension to the field of medical research in liver, heart diseases and certain forms of cancer. Most proteomics tools are also being used to study physiological and pathological events related to reproductive biology. There have been attempts to generate the proteomes of testes, sperm, seminal fluid, epididymis, oocyte, and endometrium from reproductive disease patients. Here, we have reviewed proteomics based investigations in humans over the last decade, which focus on delineating the mechanism underlying various reproductive events such as spermatogenesis, oogenesis, endometriosis, polycystic ovary syndrome, embryo development. The challenge is to harness new technologies like 2-DE, DIGE, MALDI-MS, SELDI-MS, MUDPIT, LC–MS etc., to a greater extent to develop widely applicable clinical tools in understanding molecular aspects of reproduction both in health and disease.► Proteomics studies in the field of reproductive biology ► Molecular mechanism involved in physiological and pathological conditions in reproductive tissues/body fluids in normal and diseased state ► Newer studies to solve unanswered questions associated with pathology of disease and biomarker discovery using proteomics tools

Keywords: Abbreviations; 2-DE; 2-dimensional electrophoresis; DIGE; differential-in-gel electrophoresis; MALDI-MS; matrix assisted laser desorption mass spectrometry; SELDI MS; surface enhanced desorption/ionization mass spectrometry; MUDPIT; multi-dimensional protein identification technology; LC–MS; liquid chromatography, mass spectrometry; ICAT; isotope-coded affinity tags; iTRAQ; isobaric tag for relative and absolute quantitationProteomics; Reproductive biology; Fertility; Infertility; Biomarker

Regulation of ammonium assimilation in Haloferax mediterranei: Interaction between glutamine synthetase and two GlnK proteins by Laia Pedro-Roig; Mónica Camacho; María-José Bonete (pp. 16-23).

GlnK proteins belong to the PII superfamily of signal transduction proteins and are involved in the regulation of nitrogen metabolism. These proteins are normally encoded in an operon together with the structural gene for the ammonium transporter AmtB. Haloferax mediterranei possesses two genes encoding for GlnK, specifically, glnK₁ and glnK₂. The present study marks the first investigation of PII proteins in haloarchaea, and provides evidence for the direct interaction between glutamine synthetase and both GlnK₁ and GlnK₂. Complex formation between glutamine synthetase and the two GlnK proteins is demonstrated with pure recombinant protein samples using in vitro activity assays, gel filtration chromatography and western blotting. This protein–protein interaction increases glutamine synthetase activity in the presence of 2-oxoglutarate. Separate experiments that were carried out with GlnK₁ and GlnK₂ produced equivalent results.► H. mediterranei has two PII gene copies, both attached to ammonium transporters. ► 2-oxoglutarate as a signal for GlnK interaction with glutamine synthetase. ► Glutamine synthetase activity increase due to formation of complex with GlnK.

Keywords: Abbreviations; GS; glutamine synthetase; NAGK; N-acetylglutamate kinase; ORF; open reading frame; TCA; tricarboxylic acid cycle; GS/GOGAT; glutamine synthetase/glutamate synthaseGlnK; PII; Haloferax mediterranei; Nitrogen metabolism regulation; 2-oxoglutarate; Glutamine synthetase

The effect of fulvic acid on pre‐ and postaggregation state of Aβ_17–42: Molecular dynamics simulation studies by Sharad Verma; Amit Singh; Abha Mishra (pp. 24-33).

Alzheimer's disease (AD), a neurodegenerative disorder, is directly related to the aggregation of Aβ peptides. These peptides can self-assemble from monomers to higher oligomeric or fibrillar structures in a highly ordered and efficient manner. This self-assembly process is accompanied by a structural transition of the aggregated proteins from their normal fold into a predominantly β-sheet secondary structure. 14ns molecular dynamics simulation revealed that fulvic acid interrupted the dimer formation of Aβ_17–42 peptide while in its absence Aβ_17–42 dimer formation occurred at ~12ns. Additionally, fulvic acid disrupted the preformed Aβ_17–42 trimer in a very short time interval (12ns). These results may provide an insight in the drug design against Aβ_17–42 peptide aggregation using fulvic acid as lead molecule against Aβ_17–42 mediated cytotoxicity and neurodegeneration.Antiaggregation effect of fulvic acid on Aβ peptide trimer.Display Omitted► Inhibition of Aβ dimer formation by fulvic acid evidenced by MD simulation ► Antiaggregation effect of fulvic acid on Aβ trimer evidenced by MD simulation ► Helix formation is not a mandatory step for Aβ dimer formation.

Keywords: Alzheimer's disease; Amyloid beta peptides; Fulvic acid; Molecular dynamics simulation

Spectroscopic analysis of polymerization and exonuclease proofreading by a high-fidelity DNA polymerase during translesion DNA synthesis by Babho Devadoss; Irene Lee; Anthony J. Berdis (pp. 34-45).

High fidelity DNA polymerases maintain genomic fidelity through a series of kinetic steps that include nucleotide binding, conformational changes, phosphoryl transfer, polymerase translocation, and nucleotide excision. Developing a comprehensive understanding of how these steps are coordinated during correct and pro-mutagenic DNA synthesis has been hindered due to lack of spectroscopic nucleotides that function as efficient polymerase substrates. This report describes the application of a non-natural nucleotide designated 5-naphthyl-indole-2′-deoxyribose triphosphate which behaves as a fluorogenic substrate to monitor nucleotide incorporation and excision during the replication of normal DNA versus two distinct DNA lesions (cyclobutane thymine dimer and an abasic site). Transient fluorescence and rapid-chemical quench experiments demonstrate that the rate constants for nucleotide incorporation vary as a function of DNA lesion. These differences indicate that the non-natural nucleotide can function as a spectroscopic probe to distinguish between normal versus translesion DNA synthesis. Studies using wild-type DNA polymerase reveal the presence of a fluorescence recovery phase that corresponds to the formation of a pre-excision complex that precedes hydrolytic excision of the non-natural nucleotide. Rate constants for the formation of this pre-excision complex are dependent upon the DNA lesion, and this suggests that the mechanism of exonuclease proofreading is regulated by the nature of the formed mispair. Finally, spectroscopic evidence confirms that exonuclease proofreading competes with polymerase translocation. Collectively, this work provides the first demonstration for a non-natural nucleotide that functions as a spectroscopic probe to study the coordinated efforts of polymerization and exonuclease proofreading during correct and translesion DNA synthesis.► Non-natural nucleotide that monitors incorporation and excision via fluorescence ► A nucleotide that probes the mechanism of normal versus translesion DNA synthesis ► Identification of a pre-excision complex formed with different DNA lesions ► Exonuclease proofreading is in direct competition with polymerase translocation.

Keywords: Abbreviations; gp43; wild-type bacteriophage T4 DNA polymerase; gp43exo; ⁻; exonuclease-deficient bacteriophage T4 DNA polymerase; dATP; deoxyadenosine triphosphate; dGTP; deoxyguanosine triphosphate; 2-APTP; 2-aminopurine-2′-deoxyribose triphosphate; 5-NapITP; 5-naphthyl-indole-2′-deoxyribose triphosphate; EDTA; ethylenediaminetetraacetate sodium salt; DTT; dithiothreitol; dNTP; deoxynucleoside triphosphate; Pol; DNA polymeraseDNA polymerase; Translesion DNA synthesis; Exonuclease proofreading; Non-natural nucleotide

Structure–activity relationship for enantiomers of potent inhibitors of B. anthracis dihydrofolate reductase by Christina R. Bourne; Nancy Wakeham; Baskar Nammalwar; Vladimir Tseitin; Philip C. Bourne; Esther W. Barrow; Shankari Mylvaganam; Kal Ramnarayan; Richard A. Bunce; K. Darrell Berlin; William W. Barrow (pp. 46-52).

Background: Bacterial resistance to antibiotic therapies is increasing and new treatment options are badly needed. There is an overlap between these resistant bacteria and organisms classified as likely bioterror weapons. For example, Bacillus anthracis is innately resistant to the anti-folate trimethoprim due to sequence changes found in the dihydrofolate reductase enzyme. Development of new inhibitors provides an opportunity to enhance the current arsenal of anti-folate antibiotics while also expanding the coverage of the anti-folate class. Methods: We have characterized inhibitors of B. anthracis dihydrofolate reductase by measuring the K_i and MIC values and calculating the energetics of binding. This series contains a core diaminopyrimidine ring, a central dimethoxybenzyl ring, and a dihydrophthalazine moiety. We have altered the chemical groups extended from a chiral center on the dihydropyridazine ring of the phthalazine moiety. The interactions for the most potent compounds were visualized by X-ray structure determination. Results: We find that the potency of individual enantiomers is divergent with clear preference for the S-enantiomer, while maintaining a high conservation of contacts within the binding site. The preference for enantiomers seems to be predicated largely by differential interactions with protein residues Leu29, Gln30 and Arg53. Conclusions: These studies have clarified the activity of modifications and of individual enantiomers, and highlighted the role of the less-active R-enantiomer in effectively diluting the more active S-enantiomer in racemic solutions. This directly contributes to the development of new antimicrobials, combating trimethoprim resistance, and treatment options for potential bioterrorism agents.Display Omitted ► B. anthracis DHFR is effectively inhibited by dihydrophthalazine inhibitors. ► Derivations at the chiral center of these inhibitors markedly affect the potency. ► Enzyme and whole cell inhibition and crystallography indicate best derivations. ► In silico calculations highlight role of enantiomeric energy differences. ► Potency is maximized with S-phenyl or S-isobutenyl groups on the phthalazine.

Keywords: Antibiotic resistance; Bacillus anthracis; Dihydrofolate reductase; Dihydrophthalazine; Enantiomer; Racemate

Stability of trimeric DENV envelope protein at low and neutral pH: An insight from MD study by Kshatresh Dutta Dubey; Amit Kumar Chaubey; Rajendra Prasad Ojha (pp. 53-64).

Change in pH plays a crucial role in the stability and function of the dengue envelope (DENV) protein during conformational transition from dimeric (pre-fusion state) to trimeric form (post-fusion state). In the present study we have performed various molecular dynamics (MD) simulations of the trimeric DENV protein at different pH and ionic concentrations. We have used total binding energy to justify the stability of the complex using the MMPBSA method. We found a remarkable increase in the stability of the complex at neutral pH (pH~7) due to the increment of sodium ions. However, at very low pH (pH~4), the total energy of the complex becomes high enough to destabilize the complex. At a specific pH, almost at a range of 6, the stability of the complex is significantly better than the stability of the trimer at neutral pH, which connotes that the trimer is most stable at this pH (pH~6).► Prepared three complexes at different ionic strengths at normal pH ► Performed MD simulations and MM–PBSA study ► Two trimeric complexes at different pH ► Justified stability and role of ions for a total of five complexes

Keywords: Trimeric interaction; MM–PBSA; Thermodynamical parameter; B-factor; Ionic concentration; Dengue envelope protein

Stability of early-stage amyloid-β(1–42) aggregation species by Kelley A. Coalier; Geeta S. Paranjape; Sanjib Karki; Michael R. Nichols (pp. 65-70).

Accumulation of aggregated amyloid-β protein (Aβ) is an important feature of Alzheimer's disease. There is significant interest in understanding the initial steps of Aβ aggregation due to the recent focus on soluble Aβ oligomers. In vitro studies of Aβ aggregation have been aided by the use of conformation-specific antibodies which recognize shape rather than sequence. One of these, OC antiserum, recognizes certain elements of fibrillar Aβ across a broad range of sizes. We have observed the presence of these fibrillar elements at very early stages of Aβ incubation. Using a dot blot assay, OC-reactivity was found in size exclusion chromatography (SEC)-purified Aβ(1–42) monomer fractions immediately after isolation (early-stage). The OC-reactivity was not initially observed in the same fractions for Aβ(1–40) or the aggregation-restricted Aβ(1–42) L34P but was detected within 1–2weeks of incubation. Stability studies demonstrated that early-stage OC-positive Aβ(1–42) aggregates were resistant to 4M urea or guanidine hydrochloride but sensitive to 1% sodium dodecyl sulfate (SDS). Interestingly, the sensitivity to SDS diminished over time upon incubation of the SEC-purified Aβ(1–42) solution at 4°C. Within 6–8days the OC-positive Aβ42 aggregates were resistant to SDS denaturation. The progression to, and development of, SDS resistance for Aβ(1–42) occurred prior to thioflavin T fluorescence. In contrast, Aβ(1–40) aggregates formed after 6days of incubation were sensitive to both urea and SDS. These findings reveal information on some of the earliest events in Aβ aggregation and suggest that it may be possible to target early-stage aggregates before they develop significant stability.► We observed fibrillar structural elements at very early stages of Aβ incubation. ► We compared the formation of these elements between Aβ(1–42) and Aβ(1–40). ► We identified stability differences between Aβ(1–42) and Aβ(1–40) oligomers. ► We demonstrated the development of SDS resistance in early Aβ(1–42) oligomers.

Keywords: Abbreviations; AD; Alzheimer's disease; Aβ; amyloid-β protein; GuHCl; guanidine hydrochloride; HFIP; hexafluoroisopropanol; SDS; sodium dodecyl sulfate; SEC; size exclusion chromatography; ThT; thioflavin TAmyloid-beta protein; Aggregation; Fibrillar oligomer

How phosphorylation activates the protein phosphatase-1 • inhibitor-2 complex by John F. Cannon (pp. 71-86).

Phosphorylation regulates activity of many proteins; however, atomic level details are known for very few examples. Inhibitor-2 (I2) squelches the ubiquitous protein phosphatase-1 (PP1) enzyme activity by blocking access to the metal-containing active site. I2 Thr74 phosphorylation results in PP1 activation without I2 dissociation from the PP1–I2 complex. The dynamic disordered structure of the 73-residue segment of I2 containing Thr74, prevented visualization by X-ray crystallography of PP1–I2. In this work, I generated structures of this segment using simulated annealing to NMR restraints, fused them to the crystallographic PP1–I2 coordinates, and used molecular dynamics to study the impact of Thr74 phosphorylation on structural alterations leading to PP1 activation. Frequencies of I2 Tyr149 displacement from the PP1 active site, rotation of the phenolic Tyr149 side chain to prevent its reinsertion, and repositioning the I2 inhibitory helix to expose the PP1 active site to solvent and substrates significantly increased upon I2 Thr74 phosphorylation. After these steps, a second metal bound to produce PP1–Mn₂–I2, which held the phosphorylated form of I2 to its active site less tightly than it held dephosphorylated I2. I2 Thr74 lies on the edge of variable dynamic communities of residues where it forms various allosteric pathways that induce motions at the PP1 active site 20Å away. These molecular dynamics simulations show how an unstructured region of I2 can harness enhanced rapid movements around phosphorylated Thr74 to pry I2 residues away from the PP1 active site in early steps of PP1–I2 activation.► Phosphorylation of protein phosphatase-1 (PP1) inhibitor-2 (I2) activates PP1–I2. ► The intrinsically disordered I2 phosphorylation region was modeled using NMR data. ► Molecular dynamics revealed phosphorylation induced structural alterations. ► I2 phosphorylation increased frequencies three initial steps of PP1–I2 activation. ► I2 phosphorylation-induced rapid movements were transmitted to the PP1 active site.

Keywords: Intrinsically disordered protein; Protein phosphorylation; Protein phosphatase; Molecular dynamics

Biophysical characterization of higher plant Rubisco activase by J. Nathan Henderson; Suratna Hazra; Alison M. Dunkle; Michael E. Salvucci; Rebekka M. Wachter (pp. 87-97).

Rubisco activase (Rca) is a chaperone-like protein of the AAA+ family, which uses mechano-chemical energy derived from ATP hydrolysis to release tightly bound inhibitors from the active site of the primary carbon fixing enzyme ribulose 1,5-bisphosphate oxygenase/carboxylase (Rubisco). Mechanistic and structural investigations of Rca have been hampered by its exceptional thermolability, high degree of size polydispersity and propensity towards subunit aggregation. In this work, we have characterized the thermal stability and self-association behavior of recombinant Rca preparations, and have developed ligand screening methods. Thermal denaturation profiles generated by circular dichroism indicate that creosote and tobacco short-form Rcas are the most stable proteins examined, with an estimated mid-point temperature of 45–47°C for protein denaturation. We demonstrate that ADP provides a higher degree of stabilization than ATP, that magnesium ions have a small stabilizing effect on ATP-bound, but a significant destabilizing effect on ADP-bound Rca, and that phosphate provides weak stabilization of the ADP-bound form of the protein. A dimeric species was identified by size-exclusion chromatography, suggesting that the two-subunit module may comprise the basic building block for larger assemblies. Evidence is provided that chromatographic procedures reflect non-equilibrium multimeric states. Dynamic light scattering experiments performed on nucleotide-bearing Rca support the notion that several larger, highly polydisperse assembly states coexist over a broad concentration range. No significant changes in aggregation are observed upon replacement of ADP with ATP. However, in the absence of nucleotides, the major protein population appears to consist of a monodisperse oligomer smaller than a hexamer.► ADP-complexed Rubisco activase is more thermally stabile than the ATP complex. ► Free Mg²⁺ ions have a destabilizing effect on ADP-bound, but not ATP-bound activase. ► Phosphate stabilizes the ADP-bound form. ► The direct observation of activase dimers suggests assembly from dimeric units. ► Assembly without nucleotides favors aggregation prone small oligomers.

Keywords: Abbreviations; Rubisco; ribulose-1,5-bisphosphate carboxylase/oxygenase; Rca; Rubisco activase; RuBP; ribulose bisphosphate; SE; size exclusion; HPLC; high pressure liquid chromatography; DLS; dynamic light scattering; CD; circular dichroism; Pd; polydispersity; R; _h; hydrodynamic radius; D; diffusion coefficientRubisco regulation; Protein assembly; Quaternary structure; Protein aggregation; Thermostability; Polydispersity

The loops facing the active site of prolyl oligopeptidase are crucial components in substrate gating and specificity by Zoltán Szeltner; Juhasz Tünde Juhász; Ilona Szamosi; Dean Rea; Fulop Vilmos Fülöp; Modos Károly Módos; Luiz Juliano; Polgar László Polgár (pp. 98-111).

Prolyl oligopeptidase (POP) has emerged as a drug target for neurological diseases. A flexible loop structure comprising loop A (res. 189–209) and loop B (res. 577–608) at the domain interface is implicated in substrate entry to the active site. Here we determined kinetic and structural properties of POP with mutations in loop A, loop B, and in two additional flexible loops (the catalytic His loop, propeller Asp/Glu loop). POP lacking loop A proved to be an inefficient enzyme, as did POP with a mutation in loop B (T590C). Both variants displayed an altered substrate preference profile, with reduced ligand binding capacity. Conversely, the T202C mutation increased the flexibility of loop A, enhancing the catalytic efficiency beyond that of the native enzyme. The T590C mutation in loop B increased the preference for shorter peptides, indicating a role in substrate gating. Loop A and the His loop are disordered in the H680A mutant crystal structure, as seen in previous bacterial POP structures, implying coordinated structural dynamics of these loops. Unlike native POP, variants with a malfunctioning loop A were not inhibited by a 17-mer peptide that may bind non-productively to an exosite involving loop A. Biophysical studies suggest a predominantly closed resting state for POP with higher flexibility at the physiological temperature. The flexible loop A, loop B and His loop system at the active site is the main regulator of substrate gating and specificity and represents a new inhibitor target.► The substrate gating mechanism of POP was investigated via protein engineering. ► The mutations markedly changed specificity, catalytic power and inhibitor binding. ► The interaction of loop A with the N-terminus controls substrate gating.

Keywords: Abbreviations; Abz; 2-aminobenzoyl; BNA; β-naphthylamide; CD; circular dichroism; DLS; dynamic light scattering; DSC; differential scanning calorimetry; DTT; dithiothreitol; DTNB; 5,5'-dithiobis(2-nitrobenzoic acid); EDTA; ethylenediaminetetraacetic acid; EDDnp; N-(2,4-dinitrophenyl)-ethylenediamine; MES; 4-morpholineethanesulfonic acid; MD; molecular dynamics; Ni-NTA; nickel-nitrilotriacetic acid; GSH; reduced glutathione; GSSG; oxidized glutathione; PAGE; polyacrylamide gel electrophoresis; PBS; phosphate buffered saline; PCR; polymerase chain reaction; POP; prolyl oligopeptidase; Phe(NO; ₂; ); p-nitrophenylalanine; res.; residue; SEC; size-exclusion chromatography; TEV; tobacco etch virus; T; _m; melting temperatureProlyl oligopeptidase; Protein engineering; Enzyme catalysis; Differential scanning calorimetry; Enzyme structure; X-ray crystallography

Different pressure–temperature behavior of the structured and unstructured regions of titin by Judit Somkuti; Martonfalvi Zsolt Mártonfalvi; Miklós S.Z. Kellermayer; László Smeller (pp. 112-118).

Contrary to the classical view, according to which all proteins adopt a specific folded conformation necessary for their function, intrinsically unstructured proteins (IUPs) display random-coil-like conformation under physiological conditions. We compared the structured and unstructured domains from titin, a giant protein responsible for striated-muscle elasticity. A 171-residue-long fragment (polyE) of the disordered PEVK domain, and an Ig domain (I27) with ordered structure were investigated. FTIR (Fourier transform infrared) and fluorescence spectroscopy combined with a diamond anvil cell were used for investigation of the secondary structures under wide range of pressure and temperature. PolyE preserves its disordered characteristics across the entire range of investigated pressure (0–16kbar), temperature (0–100°C), pD (3–10.5) and different solvent conditions. The detailed temperature–pressure phase diagram of titin I27 was determined. At 30°C, increasing pressure unfolds titin I27 in one step at 10.5kbar. Increasing temperature at atmospheric pressure results in two transitions. At 50°C the secondary structure is loosened and the protein transforms into a molten-globule state. At 65°C the protein completely unfolds. Unfolding is followed by aggregation at ambient pressure. Moderate pressures (>2kbar), however, can prevent the protein from aggregation. Our experiments in wide range of physical parameters revealed four different structures for I27, while the unstructured character of the PEVK fragment is insensitive to these parameters.► PolyE preserves its disordered characteristics across a wide range of conditions. ► Detailed temperature–pressure phase diagram of titin I27 was determined. ► Phase diagram of titin I27 contains molten globule and aggregated states as well. ► I27 completely unfolds at 65°C, and a molten globule state is observed at 50°C. ► Pressure unfolds titin I27 at 10.5kbar at 30°C and at 0.9kbar at 50°C.

Keywords: Titin; I27; PEVK; High pressure; Phase diagram

Artichoke and Cynar liqueur: Two (not quite) entangled proteomes by Vicente Saez; Elisa Fasoli; Alfonsina D'Amato; Simo-Alfonso Ernesto Simó-Alfonso; Pier Giorgio Righetti (pp. 119-126).

Combinatorial peptide ligand libraries (CPLLs) have been adopted to investigate the proteome of artichoke extracts, of a home-made alcoholic infusion and of the Italian Cynar liqueur. The aim of study was not only to perform the deepest investigation so far of the artichoke proteome but also to assess the genuineness of the commercial aperitif via a three-pronged attack. First, different extraction techniques have been used for the characterization of the artichoke's proteome, secondly a home-made infusion has been analyzed and finally the proteome of the commercial drink was checked. The artichoke proteome has been evaluated via prior capture with CPLLs at four different pH (2.2, 4.0, 7.2 and 9.3) values. Via mass spectrometry analysis of the recovered fractions, after elution of the captured populations in 4% boiling SDS, we could identify a total of 876 unique gene products in the artichoke extracts, 18 in the home-made infusion and no proteins at all in the Italian Cynar liqueur, casting severe doubts on the procedure stated by the manufacturer (that should be made by an infusion of artichoke leaves plus thirteen different herbs). This could be the starting point for investigating the genuineness and natural origin of commercial drinks in order to protect consumers from adulterated products.Display Omitted► The most extensive investigation on artichoke proteome: 876 unique gene products ► In a home-made infusion of artichoke leaves, 88 proteins have been detected ► In the commercial liqueur Cynar (an infusion of artichoke leaves and 13 secret herbs) no proteins could be detected ► Artichoke and Cynar liqueur: an amour impossible?

Keywords: Abbreviations; CPLL; combinatorial peptide ligand libraries; PM; ProteoMinerArtichoke; Cynar aperitif; Low abundance proteome; Combinatorial peptide ligand libraries; Mass spectrometry

OmpA can form folded and unfolded oligomers by H. Wang; K.K. Andersen; B.S. Vad; D.E. Otzen (pp. 127-136).

The monomeric outer membrane protein OmpA from Escherichia coli has long served as a model protein for studying the folding and membrane insertion of β-barrel membrane proteins. Here we report that when OmpA is refolded in limiting amounts of surfactant (close to the cmc), it has a high propensity to form folded and unfolded oligomers. The oligomers exist both in a folded and (partially) unfolded form which both dissociate under denaturing conditions. Oligomerization does not require the involvement of the periplasmic domain and is not strongly affected by ionic strength. The folded dimers can be isolated and show native-like secondary structure; they are resistant to proteolytic attack and do not dissociate in high surfactant concentrations, indicating high kinetic stability once formed. Remarkably, OmpA also forms significant amounts of higher order structures when refolding in the presence of lipid vesicles. We suggest that oligomerization occurs by domain swapping favored by the high local concentration of OmpA molecules congregating on the same micelle or vesicle. In this model, the unfolded oligomer is stabilized by a small number of intermolecular β-strand contacts and subsequently folds to a more stable state where these intermolecular contacts are consolidated in a native-like fashion by contacts between complementary β-strands from different molecules. Our model is supported by the ability of complementary fragments to associate with each other in vitro. Oligomerization is probably avoided in the cell by the presence of cellular chaperones which maintain the protein in a monomeric state.Display Omitted► OmpA refolded from urea close to the cmc forms oligomeric and monomeric species. ► The oligomers exist both in the folded and the unfolded state. ► Folded and unfolded oligomers can interconvert. ► We propose that folding occurs by β-strand mediated domain swapping. ► This occurs in lipids as well, highlighting the need for chaperoning activities.

Keywords: Abbreviations; cmc; critical micelle concentration; FL-OmpA; full-length OmpA; OmpA; outer membrane protein A; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phospho-(1′-rac-glycerol) (sodium salt); SDS-PAGE; sodium dodcyl sulfate polyacrylamide electrophoresis; TM-OmpA; transmembrane OmpAOligomer; Domain swapping; Shared micelle; Dodecyl maltoside

Apolar distal pocket mutants of yeast cytochrome c peroxidase: Hydrogen peroxide reactivity and cyanide binding of the TriAla, TriVal, and TriLeu variants by Anil K. Bidwai; Cassandra Meyen; Heather Kilheeney; Damian Wroblewski; Lidia B. Vitello; James E. Erman (pp. 137-148).

Three yeast cytochrome c peroxidase (CcP) variants with apolar distal heme pockets have been constructed. The CcP variants have Arg48, Trp51, and His52 mutated to either all alanines, CcP(triAla), all valines, CcP(triVal), or all leucines, CcP(triLeu). The triple mutants have detectable enzymatic activity at pH 6 but the activity is less than 0.02% that of wild-type CcP. The activity loss is primarily due to the decreased rate of reaction between the triple mutants and H₂O₂ compared to wild-type CcP. Spectroscopic properties and cyanide binding characteristics of the triple mutants have been investigated over the pH stability region of CcP, pH 4 to 8. The absorption spectra indicate that the CcP triple mutants have hemes that are predominantly five-coordinate, high-spin at pH 5 and six-coordinate, low-spin at pH 8. Cyanide binding to the triple mutants is biphasic indicating that the triple mutants have two slowly-exchanging conformational states with different cyanide affinities. The binding affinity for cyanide is reduced at least two orders of magnitude in the triple mutants compared to wild-type CcP and the rate of cyanide binding is reduced by four to five orders of magnitude. Correlation of the reaction rates of CcP and 12 distal pocket mutants with H₂O₂ and HCN suggests that both reactions require ionization of the reactants within the distal heme pocket allowing the anion to bind the heme iron. Distal pocket features that promote substrate ionization (basic residues involved in base-catalyzed substrate ionization or polar residues that can stabilize substrate anions) increase the overall rate of reaction with H₂O₂ and HCN while features that inhibit substrate ionization slow the reactions.Display Omitted► CcP mutants with apolar heme pockets retain detectable catalytic activity. ► CcP mutants react with H₂O₂ 10⁴- to 10⁷-times more slowly than wild-type CcP. ► Cyanide binding affinity is 10³-fold less than wild-type CcP. ► Rates of the H₂O₂ and HCN reactions correlate over 7 orders of magnitude for 11 CcP mutants.

Keywords: Abbreviations; CcP; generic abbreviation for cytochrome; c; peroxidase whatever the source; yCcP; authentic yeast cytochrome; c; peroxidase isolated from; S. cerevisiae; rCcP; recombinant cytochrome; c; peroxidase expressed in; E. coli; , the rCcP used in this study has an amino acid sequence identical to that of yCcP; CcP(MI); recombinant CcP expressed in; E. coli; with four amino acid variations compared to yCcP, a Met-Ile N-terminal extension and mutations T53I and D152G; CcP(triAla); triple mutant of rCcP with R48A/W51A/H52A; CcP(triVal); triple mutant of rCcP with R48V/W51V/H52V; CcP(triLeu); triple mutant of rCcP with R48L/W51L/H52L; CcP-I; CcP compound I; CD; circular dichroismCytochrome; c; peroxidase; Distal pocket mutant; Spectroscopic property; Hydrogen peroxide reaction; Cyanide binding

Effect of NaCl on the conformational stability of the thermophilic γ-glutamyltranspeptidase from Geobacillus thermodenitrificans: Implication for globular protein halotolerance by Andrea Pica; Irene Russo Krauss; Immacolata Castellano; Francesco La Cara; Giuseppe Graziano; Filomena Sica; Antonello Merlino (pp. 149-157).

The transpeptidation activity of γ-glutamyltranspeptidase from Geobacillus thermodenitrificans ( GthGT) is negligible and the enzyme is highly thermostable. Here we have examined the effect of concentrated NaCl solutions on structure, stability, dynamics and enzymatic activity of GthGT. The protein exhibited hydrolytic activity over a broad range of NaCl concentrations. Even at 4.0M NaCl, GthGT retained more than 90% of the initial activity and showed unaltered fluorescence emission, secondary structure and acrylamide quenching on tryptophan fluorescence. Furthermore, at 2.8M and 4.0M NaCl the temperature-induced unfolding profiles are dramatically changed with large (>20°C) positive shifts in the denaturation temperature. These features make GthGT an ideal system to be used in industrial processes that require high temperatures and high-salt environments. A general explanation of the NaCl effect by means of a statistical thermodynamic model is also provided, together with an analysis of residue distribution between protein surface and interior in 15 non-redundant families of halophilic and non-halophilic proteins. The results are in line with a comparative sequence and structural analysis between halophilic and non-halophilic γ-glutamyltranspeptidases which revealed that a major role in halotolerance should be played by solvent exposed negatively charged residues.Display Omitted► GthGT is active in the presence of 4M NaCl. ► GthGT is stabilized by the presence of NaCl. ► NaCl stabilizing effect has been studied by a statistical thermodynamic model. ► Negative charged residues on the surface are important for protein halotolerance.

Keywords: Protein stabilization; Circular dichroism; NaCl; Fluorescence; Gamma-glutamyl transferase; Gamma-glutamyl transpeptidase

The heterodimerization of platelet-derived chemokines by James Carlson; Sarah A. Baxter; Dreau Didier Dréau; Irina V. Nesmelova (pp. 158-168).

Chemokines encompass a large family of proteins that act as chemoattractants and are involved in many biological processes. In particular, chemokines guide the migration of leukocytes during normal and inflammatory conditions. Recent studies reveal that the heterophilic interactions between chemokines significantly affect their biological activity, possibly representing a novel regulatory mechanism of the chemokine activities. The co-localization of platelet-derived chemokines in vivo allows them to interact. Here, we used nano-spray ionization mass spectrometry to screen eleven different CXC and CC platelet-derived chemokines for possible interactions with the two most abundant chemokines present in platelets, CXCL4 and CXCL7. Results indicate that many screened chemokines, although not all of them, form heterodimers with CXCL4 and/or CXCL7. In particular, a strong heterodimerization was observed between CXCL12 and CXCL4 or CXCL7. Compared to other chemokines, the main structural difference of CXCL12 is in the orientation and packing of the C-terminal alpha-helix in relation to the beta-sheet. The analysis of one possible structure of the CXCL4/CXCL12 heterodimer, CXC-type structure, using molecular dynamics (MD) trajectory reveals that CXCL4 may undergo a conformational transition to alter the alpha helix orientation. In this new orientation, the alpha-helix of CXCL4 aligns in parallel with the CXCL12 alpha-helix, an energetically more favorable conformation. Further, we determined that CXCL4 and CXCL12 physically interact to form heterodimers by co-immunoprecipitations from human platelets. Overall, our results highlight that many platelet-derived chemokines are capable of heterophilic interactions and strongly support future studies of the biological impact of these interactions.► We report new platelet-derived chemokine heterodimers detected by mass spectrometry ► Several strong heterodimers were observed, including CXCL4/CXCL12 heterodimer ► The stability of CXCL4/CXCL12 heterodimer was confirmed using MD simulations ► In vivo relevance of this heterodimer was confirmed by Co-IP from human platelets ► Results strongly support future functional studies of chemokine heterodimers

Keywords: Abbreviations; MD; molecular dynamics; ESI-MS; electrospray ionization mass spectrometry; co-IP; co-immunoprecipitation; Western Blot; WB; SPR; surface plasmon resonance; M; _r; molecular weight; NMR; nuclear magnetic resonance spectroscopy; PDB; Protein Data Bank; ACN; acetonitrile; GPCR; G protein-coupled receptor; GAG; glycosaminoglycan; RMSD; root-mean-square deviationChemokines; Heterodimer; Heterophilic interactions; Mass spectrometry (MS); Co-immunoprecipitation; Platelet

Fine tuning of the active site modulates specificity in the interaction of O-acetylserine sulfhydrylase isozymes with serine acetyltransferase by Francesca Spyrakis; Paolo Felici; Alexander S. Bayden; Enea Salsi; Riccardo Miggiano; Glen E. Kellogg; Pietro Cozzini; Paul F. Cook; Andrea Mozzarelli; Barbara Campanini (pp. 169-181).

O-acetylserine sulfhydrylase (OASS) catalyzes the synthesis ofl-cysteine in the last step of the reductive sulfate assimilation pathway in microorganisms. Its activity is inhibited by the interaction with serine acetyltransferase (SAT), the preceding enzyme in the metabolic pathway. Inhibition is exerted by the insertion of SAT C-terminal peptide into the OASS active site. This action is effective only on the A isozyme, the prevalent form in enteric bacteria under aerobic conditions, but not on the B-isozyme, the form expressed under anaerobic conditions. We have investigated the active site determinants that modulate the interaction specificity by comparing the binding affinity of thirteen pentapeptides, derived from the C-terminal sequences of SAT of the closely related species Haemophilus influenzae and Salmonella typhimurium, towards the corresponding OASS-A, and towards S. typhimurium OASS-B. We have found that subtle changes in protein active sites have profound effects on protein–peptide recognition. Furthermore, affinity is strongly dependent on the pentapeptide sequence, signaling the relevance of P3–P4–P5 for the strength of binding, and P1–P2 mainly for specificity. The presence of an aromatic residue at P3 results in high affinity peptides with K_diss in the micromolar and submicromolar range, regardless of the species. An acidic residue, like aspartate at P4, further strengthens the interaction and results in the higher affinity ligand of S. typhimurium OASS-A described to date. Since OASS knocked-out bacteria exhibit a significantly decreased fitness, this investigation provides key information for the development of selective OASS inhibitors, potentially useful as novel antibiotic agents.► Changes in active sites of OASS isozymes modulate pentapeptides recognition. ► Pentapeptide affinities depends on P3–P4–P5 for strength and P1–P2 for specificity. ► Selective OASS inhibitors, potentially antibiotic agents, might be developed.

Keywords: Abbreviations; OASS; O; -acetylserine sulfhydrylase; SAT; serine acetyltransferase; OAS; O; -acetylserine; PLP; pyridoxal 5′-phosphate; CS; cysteine synthase; Hi; OASS; Haemophilus influenzae O; -acetylserine sulfhydrylase; St; OASS; Salmonella typhimurium O; -acetylserine sulfhydrylase; Hi; SAT; Haemophilus influenzae; serine acetyltransferase; St; SAT; Salmonella typhimurium; serine acetyltransferasePyridoxal 5′-phosphate; Enzyme-ligand binding; Protein–protein interaction; Sulfur assimilation pathway; Antibiotics

Studies on the regioselectivity and kinetics of the action of trypsin on proinsulin and its derivatives using mass spectrometry by Qurra-tul-Ann Afza Gardner; Hooria Younas; Muhammad Akhtar (pp. 182-190).

Human M-proinsulin was cleaved by trypsin at the R³¹R³²–E³³ and K⁶⁴R⁶⁵–G⁶⁶ bonds (B/C and C/A junctions), showing the same cleavage specificity as exhibited by prohormone convertases 1 and 2 respectively. Buffalo/bovine M-proinsulin was also cleaved by trypsin at the K⁵⁹R⁶⁰–G⁶¹ bond but at the B/C junction cleavage occurred at the R³¹R³²–E³³ as well as the R³¹–R³²E³³ bond. Thus, the human isoform in the native state, with a 31 residue connecting C-peptide, seems to have a unique structure around the B/C and C/A junctions and cleavage at these sites is predominantly governed by the structure of the proinsulin itself. In the case of both the proinsulin species the cleavage at the B/C junction was preferred (65%) over that at the C/A junction (35%) supporting the earlier suggestion of the presence of some form of secondary structure at the C/A junction. Proinsulin and its derivatives, as natural substrates for trypsin, were used and mass spectrometric analysis showed that the k_cat./ K_m values for the cleavage were most favourable for the scission of the bonds at the two junctions (1.02±0.08×10⁵s−¹M−¹) and the cleavage of the K²⁹–T³⁰ bond of M-insulin-RR (1.3±0.07×10⁵s−¹M−¹). However, the K²⁹–T³⁰ bond in M-insulin, insulin as well as M-proinsulin was shielded from attack by trypsin ( k_cat./ K_m values around 1000s−¹M−¹). Hence, as the biosynthetic path follows the sequence; proinsulin→insulin-RR→insulin, the K²⁹–T³⁰ bond becomes shielded, exposed then shielded again respectively.The graphical abstract shows the conversion of proinsulin (1) into di-arginyl insulin (2) and thence of the latter into insulin (3), highlighting in vitro shielding, from tryptic cleavage, of the K²⁹–T³⁰ bond in proinsulin (1) and insulin (3) but not in di-arginyl insulin (2).Display Omitted► Mass spectrometry quantified hierarchy of tryptic sites in proinsulin derivatives. ► K_m, k_cat., and k_cat./ K_m were obtained for tryptic sites in proinsulin derivatives. ► K²⁹–T³⁰ tryptic cleavage shielded in proinsulin/insulin, not in di-arginyl insulin. ► B/C site variation of human and buffalo proinsulin causes tryptic regioselectivity.

Keywords: Abbreviations; M-proinsulin; proinsulin containing N-terminal methionine; M-insulin-RR; insulin containing methionine at the N-terminal and two arginine residues at the C-terminal of B-chain; M-insulin-R; insulin containing methionine at the N-terminal and one arginine residue at the C-terminal of B-chain; M-insulin; insulin containing methionine at the N-terminal; (C-peptide)-KR; the conventional C-peptide sequence containing lysine and arginine at the C-terminus; R-(C-peptide)-KR; the conventional C-peptide containing arginine at N-terminus and lysine and arginine at the C-terminus; TRR-(C-peptide)-KR; the conventional C-peptide containing threonine, two arginine amino acid residues at N-terminus and lysine and arginine at the C-terminus; Des-(B30)-M-insulin; insulin containing methionine at the N-terminal and lacking threonine at the C-terminus of B-chain; Des-(B30)-insulin; standard insulin lacking threonine at the C-terminus of B-chain at position 30; Des-(B23-30)-M-insulin; insulin containing methionine at the N-terminal but lacking amino acid residues from positions 23 to 30 in B-chain; Des-(B23-30)-insulin; standard insulin lacking amino acid residues from positions 23 to 30 in B-chain; B; ²⁰; –B; ³⁰; a synthetic peptide containing human insulin B-chain sequence from residues 20–30; B; ²⁰; –B; ³⁰; –RR; a synthetic peptide containing human insulin B-chain sequence from residues 20–30, and two arginine residues at the C-terminus of a peptide; PC1; prohormone convertase 1; PC2; prohormone convertase 2, MTP, microtitre plate; IS; ion source; Ref; reflectorComparative kinetics by mass spectrometry and HPLC; Proinsulin kinetic parameter; Conversion of proinsulin to insulin; Human di-arginyl insulin; Des-(B30)-insulin; Bovine/buffalo proinsulin

S-nitrosylation of Mycobacterium tuberculosis tyrosine phosphatase A (PtpA) induces its structural instability by Camila Matiollo; Gabriela Ecco; Angela Camila Orbem Menegatti; Guilherme Razzera; Javier Vernal; Hernán Terenzi (pp. 191-196).

S-nitrosylation is associated with signal transduction and microbicidal activity of nitric oxide (NO). We have recently described the S-nitrosylation of Mycobacterium tuberculosis protein tyrosine phosphatase A, PtpA, an enzyme that plays an important role in mycobacteria survival inside macrophages. This post-translational modification decreases the activity of the enzyme upon modification of a single Cys residue, C53. The aim of the present work was the investigation of the effect of S-nitrosylation in PtpA kinetic parameters, thermal stability and structure. It was observed that the K_M of nitrosylated PtpA was similar to its unmodified form, but the V_max was significantly reduced. In contrast, treatment of PtpA C53A with GSNO, did not alter either K_M or V_max. These results confirmed that PtpA S-nitrosylation occurs specifically in the non-catalytic C53 and that this modification does not affect substrate affinity. Using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy techniques it was shown that PtpA S-nitrosylation decreased protein thermal stability and promoted a local effect in the surroundings of the C53 residue, which interfered in both protein stability and function.Using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy techniques we show that S-nitrosylation in PtpA promotes a local effect in the surroundings of the C53 residue which interferes in both protein stability and function. This is the first evidence of S-nitrosylation modification related to tyrosine phosphatase stability, in contrast to those already described in the literature for other tyrosine phosphatases, where the catalytic cysteine is modified and affects the activity of the enzyme.Display Omitted► S-nitrosylation of PtpA in the non-catalytic C53 decreases its thermal stability. ► PtpA S-nitrosylation promotes structural-functional effects. ► The S-nitrosylation of PtpA affects kcat but not K_M.

Keywords: Tyrosine phosphatase A; Mycobacterium tuberculosis; Circular dichroism; S-nitrosylation

A Mn(II)–Mn(II) center in human prolidase by Roberta Besio; Maria Camilla Baratto; Roberta Gioia; Enrico Monzani; Stefania Nicolis; Lucia Cucca; Antonella Profumo; Luigi Casella; Riccardo Basosi; Ruggero Tenni; Antonio Rossi; Antonella Forlino (pp. 197-204).

Human prolidase, the enzyme responsible for the hydrolysis of the Xaa-Pro/Hyp peptide bonds, is a key player in the recycling of imino acids during the final stage of protein catabolism and extracellular matrix remodeling. Its metal active site composition corresponding to the maximal catalytic activity is still unknown, although prolidase function is of increasing interest due to the link with carcinogenesis and mutations in prolidase gene cause a severe connective tissue disorder. Here, using EPR and ICP-MS on human recombinant prolidase produced in Escherichia coli (hRecProl), the Mn(II) ion organized in a dinuclear Mn(II)–Mn(II) center was identified as the protein cofactor. Furthermore, thermal denaturation, CD/fluorescence spectroscopy and limited proteolysis revealed that the Mn(II) is required for the proper protein folding and that a protein conformational modification is needed in the transition from apo- to Mn(II)loaded-enzyme. The collected data provided a better knowledge of the human holo-prolidase and, although limited to the recombinant enzyme, the exact identity and organization of the metal cofactor as well as the conformational change required for activity were proven.► Apoprolidase was generated. ► Mn(II) was proven to be the cofactor necessary for prolidase activity. ► A dinuclear Mn(II)–Mn(II) center was identified in active prolidase. ► The Mn(II) ion was demonstrated to be necessary for prolidase structural integrity. ► A conformational modification was detected in the transition apo/holoenzyme.

Keywords: Abbreviations; ECM; extracellular matrix; ICP-MS; inductively coupled plasma mass spectrometry; XANES; X-ray absorption near edge structure; DTT; dithiothreitol; GSH; reduced glutathioneProlidase; Metallo-enzyme; Mn(II)–Mn(II) cluster; Apoenzyme

Evaluation of structural features in fungal cytochromes P450 predicted to rule catalytic diversification by Peter Hlavica (pp. 205-220).

Fungi belong to the large kingdom of lower eukaryotic organisms encompassing yeasts along with filamentous and dimorphic members. Microbial P450 enzymes have contributed to exploration of and adaptation to diverse ecological niches such as conversion of lipophilic compounds to more hydrophilic derivatives or degradation of a vast array of environmental toxicants. To better understand diversification of the catalytic behavior of fungal P450s, detailed insight into the molecular machinery steering oxidative attack on the distinctly structured endogenous and xenobiotic substrates is of preeminent interest. Based on a general, CYP102A1-related template the bulk of predicted substrate/inhibitor-binding determinants were shown to cluster near the distal heme face within the six known substrate recognition sites (SRSs) made up by the α-helical B′/F/G/I tetrad, the B′–C interhelical loop and strands of the β6-sheet, population density being highest in the structurally flexible SRS-1 and SRS-4 domains, showing a low degree of conservation. Reactivity toward ligands favorably coincides with the lipophilicity/hydrophilicity profile and bulkiness of critical amino acids acting as selective filters. Some decisive elements may also serve in maintenance of catalytic competence via their action as gatekeepers directing substrate access/positioning or stabilizers of the heme environment enabling dioxygen activation. Non-SRS residues seem to control spin state equilibria and attract redox partners by electrostatic forces. Of note, the inhibitory potency of azole-type fungicides is likely to arise from perturbation of the complex interplay of the mechanistic principles addressed above. Knowledge-supported exploitation of the topological data will be helpful in the manufacture of commodity/specialty chemicals as well as therapeutic agents. Also, engineered fungal P450s may be used to improve pollutant-specific bioremediation of contaminated soils.► Structure-function analysis was carried out with fungal P450s. ► Key determinants governing interactions with substrates, inhibitors and redox partners were elucidated. ► A generalized molecular model of crucial sites was created on the basis of the bacterial CYP102A1 template. ► Biotechnological exploitation of the topological data in medicinal and commercial areas is discussed.

Keywords: Abbreviations; AT; aminotetralines; BFZ; bifonazole; BH; benzoheterocycles; BP; benzopyrones; CHARMM; Chemistry at HARvard Macromolecular Mechanics; CoMFA; comparative molecular field analysis; CPR; NADPH-cytochrome P450 oxidoreductase; CR; chromanes; CTZ; clotrimazole; CYPED; CYP Engineering Database; DCZ; diniconazole; 5,8-DiHODE; 5,8-dihydroxyoctadecadienoic acid; DHOMST; dihydro-; O; -methylsterigmatocystin; FCZ; fluconazole; 8-HPODE; 8(; R; )-hydroperoxyoctadecadienoic acid; ICZ; itraconazole; IQ; isoquinolines; KCZ; ketoconazole; MIC; minimal inhibitory concentration; MMFF; Merck molecular force field; MOE; molecular operating environment; OMST; O; -methylsterigmatocystin; P450 (CYP); cytochrome P450; PAHs; polycyclic aromatic hydrocarbons; PCZ; posaconazole; SRS; substrate recognition site; SYBYL; molecular modeling from sequence through lead optimization; TDF; triadimefon; TDM; triadimenol; VCZ; voriconazoleFungal P450; Structure–function analysis; Substrate/inhibitor docking; Molecular modeling; Biotechnological exploitation

Stereochemical features of the envelope protein Domain III of dengue virus reveals putative antigenic site in the five-fold symmetry axis by R.O.S. Soares; A. Caliri (pp. 221-230).

We bring to attention a characteristic parasitic pattern present in the dengue virus: it undergoes several intensive thermodynamic variations due to host environmental changes, from a vector's digestive tract, through the human bloodstream and intracellular medium. Comparatively, among the known dengue serotypes, we evaluate the effects that these medium variations may induce to the overall structural characteristics of the Domain III of the envelope (E) protein, checking for stereochemical congruences that could lead to the identification of immunologic relevant regions. We used molecular dynamics and principal component analysis to study the protein in solution, for all four dengue serotypes, under distinct pH and temperature. We stated that, while the core of Domain III is remarkably rigid and effectively unaffected by most of the mentioned intensive variations, the loops account for major and distinguishable flexibilities. Therefore, the rigidity of the Domain III core provides a foothold that projects specifically two of these high flexible loop regions towards the inner face of the envelope pores, which are found at every five-fold symmetry axis of the icosahedron-shaped mature virus. These loops bear a remarkable low identity though with high occurrence of ionizable residues, including histidines. Such stereochemical properties can provide very particular serotype-specific electrostatic surface patterns, suggesting a viral fingerprint region, on which other specific molecules and ions can establish chemical interactions in an induced fit mechanism. We assert that the proposed regions share enough relevant features to qualify for further immunologic and pharmacologic essays, such as target peptide synthesis and phage display using dengue patients' sera.► We deal with dengue mechanisms of infection through molecular dynamics. ► Specific regions of the Domain III are very flexible, with fingerprint-like features. ► These loops compose envelope pores, are coordinated and suggest induced fit behavior. ► This point towards sites of relevance in the immunogenicity of dengue. ► Successful molecular design to this sites could probably halt infection stages.

Keywords: Dengue virus; Domain III; Molecular dynamics; Putative epitope; Surface pore; Induced fit

Virulence factors are released in association with outer membrane vesicles of Pseudomonas syringae pv. tomato T1 during normal growth by Chiranjit Chowdhury; Medicharla Venkata Jagannadham (pp. 231-239).

Outer membrane vesicles (OMVs) are released from Pseudomonas syringae pv. tomato T1 ( Pst T1) during their normal growth. These extracellular compartments are comprised of a complete set of biological macromolecules that includes proteins, lipids, lipopolysaccharides, etc. It is evident from proteomics analyses the OMVs of Pst T1 contain membrane- and virulence-associated proteins. In addition, OMVs of this organism are also associated with phytotoxin, coronatine. Therefore, OMVs of Pst T1 must play a significant role during pathogenicity to host plant. However, further studies are required whether these structures can serve as “vehicles” for the transport of virulence factors into the host membrane.► Outer membrane vesicles (OMVs) of Pseudomonas syringae pv. tomato T1 were characterized by biochemical analyses and mass spectrometry. ► The observed results demonstrated the structural features and physiological aspects of vesicles. ► The presence of virulence factors associated with vesicles predicted the possible involvement of OMVs in pathogenesis.

Keywords: Mass spectrometry; Outer membrane vesicle; P. syringae; Plant pathogen; Tomato plant

Expression and characterization of PhzE from P. aeruginosa PAO1: aminodeoxyisochorismate synthase involved in pyocyanin and phenazine-1-carboxylate production by Justin E. Culbertson; Michael D. Toney (pp. 240-246).

PhzE from Pseudomonas aeruginosa catalyzes the first step in the biosynthesis of phenazine-1-carboxylic acid, pyocyanin, and other phenazines, which are virulence factors for Pseudomonas species. The reaction catalyzed converts chorismate into aminodeoxyisochorismate using ammonia supplied by a glutamine amidotransferase domain. It has structural and sequence homology to other chorismate-utilizing enzymes such as anthranilate synthase, isochorismate synthase, aminodeoxychorismate synthase, and salicylate synthase. Like these enzymes, it is Mg2⁺ dependent and catalyzes a similar S_N2" nucleophilic substitution reaction. PhzE catalyzes the addition of ammonia to C2 of chorismate, as does anthranilate synthase, yet unlike anthranilate synthase it does not catalyze elimination of pyruvate from enzyme-bound aminodeoxyisochorismate. Herein, the cloning of the phzE gene, high level expression of active enzyme in E. coli, purification, and kinetic characterization of the enzyme is presented, including temperature and pH dependence. Steady-state kinetics give K_chorismate=20±4μM, K_Mg2⁺=294±22μM, K_L-gln=11±1mM, and k_cat=2.2±0.2s−¹ for a random kinetic mechanism. PhzE can use NH₄⁺ as an alternative nucleophile, while Co2⁺ and Mn2⁺ are alternative divalent metals.

Keywords: Abbreviations; AS; anthranilate synthase; SS; salicylate synthase; ADCS; aminodeoxychorismate synthase; IS; isochorismate synthase; CM; chorismate mutase; ADIC; aminodeoxyisochorismate; DHHA; trans; -2,3-dihydro-3-hydroxyanthranilic acid; GDH; glutamate dehydrogenase; LDH; lactate dehydrogenase; DON; 6-diazo-5-oxo-; l; -norleucine; GATase1; glutamine amidotransferase domainsPhzE; Pseudomonas aeruginosa; Chorismic acid; Pyocyanin; Aminodeoxyisochorismate

Recent advances in the structural mechanisms of DNA glycosylases by Sonja C. Brooks; Suraj Adhikary; Emily H. Rubinson; Brandt F. Eichman (pp. 247-271).

DNA glycosylases safeguard the genome by locating and excising a diverse array of aberrant nucleobases created from oxidation, alkylation, and deamination of DNA. Since the discovery 28years ago that these enzymes employ a base flipping mechanism to trap their substrates, six different protein architectures have been identified to perform the same basic task. Work over the past several years has unraveled details for how the various DNA glycosylases survey DNA, detect damage within the duplex, select for the correct modification, and catalyze base excision. Here, we provide a broad overview of these latest advances in glycosylase mechanisms gleaned from structural enzymology, highlighting features common to all glycosylases as well as key differences that define their particular substrate specificities.► Recent structural and mechanistic studies of DNA glycosylases are reviewed. ► DNA glycosylases excise oxidized, alkylated, or deaminated nucleobases. ► Five structural folds use base flipping to recognize and remove damage. ► A sixth architecture traps destabilized base pairs without a base binding pocket.

Keywords: Base excision repair; DNA glycosylase; Protein–DNA interactions; DNA oxidation; DNA alkylation; Active DNA demethylation

A contaminant trypsin-like activity from the timothy grass pollen is responsible for the conflicting enzymatic behavior of the major allergen Phl p 1 by Danielle Baeyens-Volant; Nasiha M'Rabet; Rachida El Mahyaoui; Ruddy Wattiez; Mohamed Azarkan (pp. 272-283).

We intend to solve whether or not Phl p 1 can be regarded as a protease. A group reported that Phl p 1 has papain-like properties and later on, that this allergen resembles cathepsin B, while another one demonstrated that Phl p 1 lacks proteinase activity and suggested that the measured activity may rise either from a recombinant Phl p 1 contaminant or as a result of an incompletely purified natural allergen. A third group reported Phl p 1 to act by a non-proteolytic activity mechanism. We report the purification of the natural Phl p 1 by means of hydrophobic interaction, gel filtration and STI-Sepharose affinity chromatographies. The Phl p 1 purity was assessed by silver-stained SDS-PAGE and by ‘in-gel’ and ‘gel-free’ approaches associated to mass spectrometry analyses. The proteolytic activity was measured using Boc–Gln–Ala–Arg–AMC and Z-Phe–Arg–AMC as substrates. While amidolytic activity could be measured with Phl p 1 after rechromatography on gel filtration, it however completely disappeared after chromatography on STI-Sepharose. The contaminant activity co-eluting with Phl p 1 was not affected by cysteine proteases inhibitors and other thiol-blocking agents, by metalloproteases inhibitors and by aspartic proteases inhibitors. However, it was completely inhibited by low molecular weight and proteinaceous serine proteases inhibitors. TLCK, but not TPCK, inhibited the contaminant activity, showing a trypsin-like behavior. The pH and temperature optimum were 8.0 and 37°C, respectively. These data indicated that Phl p 1 is not a protease. The contaminant trypsin-like activity should be considered when Phl p 1 allergenicity is emphasized.► We model two hospitals which have regulated prices and compete on quality. ► We examine changes in the level of information about hospital quality. ► Increasing information will increase quality if hospital costs are similar. ► Increasing information will decrease quality if hospital costs are very different. ► Welfare effects depend on ex-ante or ex-post assumptions about quality information.

Keywords: Phleum pratense; Group-1 allergen; β-expansin; Serine protease; Phl p 1

Crystal structure of the C-terminal domain of Mu phage central spike and functions of bound calcium ion by Kenichi Harada; Eiki Yamashita; Atsushi Nakagawa; Takamitsu Miyafusa; Kouhei Tsumoto; Takashi Ueno; Yoshiharu Toyama; Shigeki Takeda (pp. 284-291).

Bacteriophage Mu, which has a contractile tail, is one of the most famous genus of Myoviridae. It has a wide host range and is thought to contribute to horizontal gene transfer. The Myoviridae infection process is initiated by adhesion to the host surface. The phage then penetrates the host cell membrane using its tail to inject its genetic material into the host. In this penetration process, Myoviridae phages are proposed to puncture the membrane of the host cell using a central spike located beneath its baseplate. The central spike of the Mu phage is thought to be composed of gene 45 product (gp45), which has a significant sequence homology with the central spike of P2 phage (gpV). We determined the crystal structure of shortened Mu gp45Δ1-91 (Arg92–Gln197) at 1.5Å resolution and showed that Mu gp45 is a needlelike structure that punctures the membrane. The apex of Mu gp45 and that of P2 gpV contained iron, chloride, and calcium ions. Although the C-terminal domain of Mu gp45 was sufficient for binding to the E. coli membrane, a mutant D188A, in which the Asp amino acid residue that coordinates the calcium ion was replaced by Ala, did not exhibit a propensity to bind to the membrane. Therefore, we concluded that calcium ion played an important role in interaction with the host cell membrane.► The central spike of the Mu phage is composed of gene 45 product (gp45). ► We determined the crystal structure of shortened gp45D1-91 to 1.5 Å resolution. ► Three identical polypeptides formed a triangular prism. ► The bound calcium ion played an important role in interaction with the host cell.

Keywords: Abbreviations; DSC; differential scanning calorimetry; EDTA; ethylenediaminetetraacetic acid; gp45Δ1-63, gp45Δ1-91, gp45Δ1-99; the recombinant C-terminal fragment of Mu phage gp45 corresponding to Ser64–Gln197, Arg92–Gln197, and Glu100–Gln197, respectively, fused to a histidine tag; PCR; polymerase chain reaction; PEG; polyethylene glycol; MAD; multi wavelength anomalous dispersion; MES; 2-morpholinoethanesulfonic acid; QCM; a quartz crystal microbalance; rmsd; root mean square deviations; SAM; self-assembling monolayer; Tris; tris(hydroxymethyl)aminomethaneX-ray crystallography; Bacteriophage; Tail spike; Triple β-helix; Cell adsorption

Formation of the death domain complex between FADD and RIP1 proteins in vitro by Young-Hoon Park; Mi Suk Jeong; Hyun Ho Park; Se Bok Jang (pp. 292-300).

Fas-associated death domain (FADD) protein is an adapter molecule that bridges the interactions between membrane death receptors and initiator caspases. The death receptors contain an intracellular death domain (DD) which is essential to the transduction of the apoptotic signal. The kinase receptor-interacting protein 1 (RIP1) is crucial to programmed necrosis. The cell type interplay between FADD and RIP1, which mediates both necrosis and NF-κB activation, has been evaluated in other studies, but the mechanism of the interaction of the FADD and RIP1 proteins remain poorly understood. Here, we provided evidence indicating that the DD of human FADD binds to the DD of RIP1 in vitro. We developed a molecular docking model using homology modeling based on the structures of FADD and RIP1. In addition, we found that two structure-based mutants (G109A and R114A) of the FADD DD were able to bind to the RIP1 DD, and two mutations (Q169A and N171A) of FADD DD and four mutations (G595, K596, E620, and D622) of RIP1 DD disrupted the FADD–RIP1 interaction. Six mutations (Q169A, N171A, G595, K596, E620, and D622) lowered the stability of the FADD–RIP1 complex and induced aggregation that structurally destabilized the complex, thus disrupting the interaction.► DD of human FADD binds to the DD of RIP1 in vitro. ► Structural models of FADD DD and RIP1 DD interactions were predicted. ► Six mutations of FADD or RIP1 disrupted FADD and RIP1 interaction.

Keywords: FADD; RIP1; Modeling; Interaction

Binding of bivalent ions to actinomycete mannanase is accompanied by conformational change and is a key factor in its thermal stability by Yuya Kumagai; Kayoko Kawakami; Misugi Uraji; Tadashi Hatanaka (pp. 301-307).

The study aimed to define the key factors involved in the modulation of actinomycete mannanases. We focused on the roles of carbohydrate-binding modules (CBMs) and bivalent ions. To investigate the effects of these factors, two actinomycete mannanase genes were cloned from Streptomyces thermoluteus (StManII) and Streptomyces lividans (SlMan). CBMs fused to mannanase catalytic domains do not affect the thermal stability of the proteins. CBM2 of StManII increased the catalytic efficiency toward soluble-mannan and insoluble-mannan by 25%–36%, and CBM10 of SlMan increased the catalytic efficiency toward soluble-mannan by 40%–50%. Thermal stability of wild-type and mutant enzymes was enhanced by calcium and manganese. Thermal stability of SlMandC was also slightly enhanced by magnesium. These results indicated that bivalent ion-binding site responsible for thermal stability was in the catalytic domains. Thermal stability of mannanase differed in the kinds of bivalent ions. Isothermal titration calorimetry revealed that the catalytic domain of StManII bound bivalent ions with a K_a of 5.39±0.45×10³–7.56±1.47×10³M−¹, and the catalytic domain of SlMan bound bivalent ions with a K_a of 1.06±0.34×10³–3.86±0.94×10³M−¹. The stoichiometry of these bindings was consistent with one bivalent ion-binding site per molecule of enzyme. Circular dichroism spectrum revealed that the presence of bivalent ions induced changes in the secondary structures of the enzymes. The binding of certain bivalent ion responsible for thermal stability was accompanied by a different conformational change by each bivalent ion. Actinomycete mannanases belong to GHF5 which contained various hemicellulases; therefore, the information obtained from mannanases applies to the other enzymes.► We cloned and expressed the mannanases of Streptomyces thermoluteus and S. lividans. ► Carbohydrate-binding modules enhanced mannanase activity, but not thermal stability. ► Binding of certain bivalent ions to mannanase enhanced thermal stability. ► Binding involved positive entropy change due to dehydration of protein-bivalent ion. ► Conformational change induced by certain bivalent ions was different among mannanases.

Keywords: Abbreviations; ANS; 1-anilinonaphthalene-8-sulfonic; CBM; carbohydrate-binding module; CD; circular dichroism; DNS; 3,5-dinitrosalicylic acid; GM; konjac glucomannan; ITC; isothermal titration calorimetry; IvMan; ivory nut mannan; LBG; locust bean gum; PIPES; piperazine-; N; ,; N; ′-bis(2-ethanesulfonic acid); StManII; Streptomyces thermoluteus; mannanase; StManIIdC; catalytic domain of StManII; SlMan; Streptomyces lividans; mannanase; SlMandC; catalytic domain of SlManMannanase; Actinomycete; Calcium; Thermal stability; Glycoside hydrolase family 5; Conformational change

Site-specific structure of Aβ(25–35) peptide: Isotope-assisted vibrational circular dichroism study by Ganesh Shanmugam; Prasad L. Polavarapu (pp. 308-316).

We investigated the site-specific local structure of an amyloid peptide, NH₂–GSNKGAIIGLM–COOH [Aβ(25–35)], one of the active fragments of amyloid β peptide that is known to be responsible for Alzheimer's disease, in the fibrillar aggregated state. Isotope-assisted infrared vibrational circular dichroism (VCD) and absorption (VA) spectroscopy were used for the parent Aβ(25–35) peptide, along with doubly¹³C labeled peptides at the carbonyl groups of residues 29 (Gly) and 30 (Ala) [Aβ(25–35:¹³C-29/30)] and at the carbonyl groups of residues 33 (Gly) and 34 (Leu) [Aβ(25–35:¹³C-33/34)]. The present results confirm that Aβ(25–35) peptide fibrils adopt a β-sheet structure and isotopic dilution experiments suggest a parallel β-sheet structure. The isotopic shifts suggest that the microenvironment of residues 29 (Gly) and 30 (Ala) could be different from that of residues 33 (Gly) and 34 (Leu). An unusual enhancement for the amide II′ VCD intensities of Aβ(25–35:¹³C-29/30) and Aβ(25–35:¹³C-33/34) peptide fibrils, considered to originate from inter-strand coupling, was found for the first time. The structural information reported in this manuscript has important implications in understanding the role of this peptide in the development of Alzheimer's disease.Display Omitted► Investigated the structure of Aβ(25–35) peptide in fibrillar aggregated state. ► Used vibrational circular dichroism and absorption spectra of¹³C isotope labels. ► Isotope labeled study indicates aperiodic N-terminus. ► Isotope dilution study indicates parallel beta sheet structure. ► The inter-strand coupling enhances the intensity in the amide II′ region.

Keywords: Amyloid; Beta sheet; Vibrational; Circular dichroism; Infrared; Isotope

Energy based pharmacophore mapping of HDAC inhibitors against class I HDAC enzymes by Subha Kalyaanamoorthy; Yi-Ping Phoebe Chen (pp. 317-328).

Histone deacetylases (HDACs) are important class of enzymes that deacetylate the ε-amino group of the lysine residues in the histone tails to form a closed chromatin configuration resulting in the regulation of gene expression. Inhibition of these HDACs enzymes have been identified as one of the promising approaches for cancer treatment. The type-specific inhibition of class I HDAC enzymes is known to elicit improved therapeutic effects and thus, the search for promising type-specific HDAC inhibitors compounds remains an ongoing research interest in cancer drug discovery. Several different strategies are employed to identify the features that could identify the isoform specificity factors in these HDAC enzymes. This study combines the insilico docking and energy-optimized pharmacophore (e-pharmacophore) mapping of several known HDACi's to identify the structural variants that are significant for the interactions against each of the four class I HDAC enzymes. Our hybrid approach shows that all the inhibitors with at least one aromatic ring in their linker regions hold higher affinities against the target enzymes, while those without any aromatic rings remain as poor binders. We hypothesize the e-pharmacophore models for the HDACi's against all the four Class I HDAC enzymes which are not reported elsewhere. The results from this work will be useful in the rational design and virtual screening of more isoform specific HDACi's against the class I HDAC family of proteins.► Aromatic ring in the HDAC inhibitor linker show higher binding affinity. ► E-pharmacophore model unfolds the structures that contribute to sizable binding. ► Hydrophobic interaction at linker-cap junction is important for HDAC1 & 2 binding. ► A donor at cap, hbonding with ASP101 is the key interaction in HDAC8 binding. ► A donor at cap, hbonding with PHE199 is key entity for HDAC3 binding.

Keywords: Abbreviations; HDAC; Histone Deacetylase; HDACi; Histone Deacetylase Inhibitors; ZBG; Zinc Binding Groupe-Pharmacophore; HDAC; HDAC inhibitors; structure based descriptors

Key aromatic residues at subsites +2 and +3 of glycoside hydrolase family 31 α-glucosidase contribute to recognition of long-chain substrates by Takayoshi Tagami; Masayuki Okuyama; Hiroyuki Nakai; Young-Min Kim; Haruhide Mori; Kazunori Taguchi; Birte Svensson; Atsuo Kimura (pp. 329-335).

Glycoside hydrolase family 31 α-glucosidases (31AGs) show various specificities for maltooligosaccharides according to chain length. Aspergillus niger α-glucosidase (ANG) is specific for short-chain substrates with the highest k_cat/ K_m for maltotriose, while sugar beet α-glucosidase (SBG) prefers long-chain substrates and soluble starch. Multiple sequence alignment of 31AGs indicated a high degree of diversity at the long loop (N-loop), which forms one wall of the active pocket. Mutations of Phe236 in the N-loop of SBG (F236A/S) decreased k_cat/ K_m values for substrates longer than maltose. Providing a phenylalanine residue at a similar position in ANG (T228F) altered the k_cat/ K_m values for maltooligosaccharides compared with wild-type ANG, i.e., the mutant enzyme showed the highest k_cat/ K_m value for maltotetraose. Subsite affinity analysis indicated that modification of subsite affinities at +2 and +3 caused alterations of substrate specificity in the mutant enzymes. These results indicated that the aromatic residue in the N-loop contributes to determining the chain-length specificity of 31AGs.► Phe236 in the N-loop of SBG is involved in longer-maltooligosaccharide specificity. ► Mutation of Phe236 lowered subsite affinities at +2 and +3. ► Giving the N-loop of ANG a Phe residue somewhat altered substrate specificity. ► The Phe residue in the N-loop of ANG raised the subsite affinity at +3. ► Phe residue in the N-loop implicates the chain-length specificity in GH31AGs.

Keywords: Abbreviations; ANG; Aspergillus niger; α-glucosidase; AOX; 1; alcohol oxidase 1 gene; BWG; buckwheat α-glucosidase; CtMGAM; C-terminal subunit of human maltase-glucoamylase; DP; degree of polymerization; GAP; glyceraldehyde-3-phosphate dehydrogenase gene; Gn; maltooligosaccharides; G2–G7; Gn with DP; =; 2–7, respectively; G18; maltodextrin with average DP; =; 18; NtMGAM; N-terminal subunit of human maltase-glucoamylase; rANG; recombinant; A. niger; α-glucosidase; rSBG; recombinant sugar beet α-glucosidase; SBG; sugar beet α-glucosidase; 31AG; glycoside hydrolase family 31 α-glucosidase; 3D; three-dimensionalα-Glucosidase; Glycoside hydrolase family 31; Substrate specificity; Subsite affinity; Aromatic residue

In-situ spectroscopic investigation of ultrasonic assisted unfolding and aggregation of insulin by Helge Pfeiffer; Nikos Chatziathanasiou; Bert Verstraeten; Filip Meersman; Christ Glorieux; Karel Heremans; Martine Wevers (pp. 336-341).

It is well-known that fibrillogenesis of proteins can be influenced by diverse external parameters, such as temperature, pressure, agitation or chemical agents. The present preliminary study suggests that ultrasonic excitation at moderate intensities has a significant influence on the unfolding and aggregation behaviour of insulin. Irradiation with an average sound intensity of even as low as 70mW/cm² leads to a lowering of the unfolding and aggregation temperature up to 7K. The effect could be explained by an increase of the aggregation kinetics due to ultrasonically induced acoustic micro-streaming in the insulin solution that most probably enhances the aggregation rate. The clear and remarkable effect at relatively low sound intensities offers interesting options for further applications of ultrasound in biophysics and biochemistry. On the other hand, a process that causes a change of kinetics equivalent to 7K also gives a warning signal concerning the safety of those medical ultrasonic devices that work in this intensity range.Display Omitted► An ultrasonic actuator cell was developed to insonify insulin solutions at different temperatures. ► FTIR‐spectroscopy was used to measure denaturation/aggregation temperature of insulin. ► Ultrasonic irradiation reduces the transition temperature for insulin denaturation/aggregation.

Keywords: Fibrillogenesis; Insulin; Ultrasound; Fourier transform infrared spectroscopy; Atomic force microscopy; Aggregation

Structural disorder and local order of hNopp140 by Agnes Tantos; Kriszta Szrnka; Beata Szabo; Monika Bokor; Pawel Kamasa; Peter Matus; Angela Bekesi; Kalman Tompa; Kyou-Hoon Han; Peter Tompa (pp. 342-350).

Human nucleolar phosphoprotein p140 (hNopp 140) is a highly phosphorylated protein inhibitor of casein kinase 2 (CK2). As in the case of many kinase-inhibitor systems, the inhibitor has been described to belong to the family of intrinsically disordered proteins (IDPs), which often utilize transient structural elements to bind their cognate enzyme. Here we investigated the structural status of this protein both to provide distinct lines of evidence for its disorder and to point out its transient structure potentially involved in interactions and also its tendency to aggregate. Structural disorder of hNopp140 is apparent by its anomalous electrophoretic mobility, protease sensitivity, heat stability, hydrodynamic behavior on size-exclusion chromatography,¹H NMR spectrum and differential scanning calorimetry scan. hNopp140 has a significant tendency to aggregate and the change of its circular dichroism spectrum in the presence of 0–80% TFE suggests a tendency to form local helical structures. Wide-line NMR measurements suggest the overall disordered character of the protein. In all, our data suggest that this protein falls into the pre-molten globule state of IDPs, with a significant tendency to become ordered in the presence of its partner as demonstrated in the presence of transcription factor IIB (TFIIB).► hNopp140 belongs to the premolten globule class of IDPs. ► hNopp140 uses pre-structured motifs to form diverse interactions. ► hNopp140 binding to TFIIB involves the elevation of helical content. ► The diverse cellular functions of hNopp140 stem from its disordered state. ► The high level of phosphorylation of hNopp140 is linked to its disordered nature.

Keywords: Intrinsically disordered protein; Human nucleolar phosphoprotein p140; Pre-structured motif; Local structure

Structural and functional studies of Leishmania braziliensis Hsp90 by K.P. Silva; T.V. Seraphim; J.C. Borges (pp. 351-361).

The ubiquitous Hsp90 is critical for protein homeostasis in the cells, stabilizing “client” proteins in a functional state. Hsp90 activity depends on its ability to bind and hydrolyze ATP, involving various conformational changes that are regulated by co-chaperones, posttranslational modifications and small molecules. Compounds like geldanamycin (GA) and radicicol inhibit the Hsp90 ATPase activity by occupying the ATP binding site, which can lead client protein to degradation and also inhibit cell growth and differentiation in protozoan parasites. Our goal was to produce the recombinant Hsp90 of Leishmania braziliensis (LbHsp90) and construct of its N-terminal (LbHsp90N) and N-domain and middle-domain (LbHsp90NM), which lacks the C-terminal dimerization domain, in order to understand how Hsp90 works in protozoa. The recombinant proteins were produced folded as attested by spectroscopy experiments. Hydrodynamic experiments revealed that LbHsp90N and LbHsp90NM behaved as elongated monomers while LbHsp90 is an elongated dimer. All proteins prevented the in vitro citrate synthase and malate dehydrogenase aggregation, attesting that they have chaperone activity, and interacted with adenosine ligands with similar dissociation constants. The LbHsp90 has low ATPase activity (k_cat=0.320min−¹) in agreement with Hsp90 orthologs, whereas the LbHsp90NM has negligible activity, suggesting the importance of the dimeric protein for this activity. The GA interacts with LbHsp90 and with its domain constructions with different affinities and also inhibits the LbHsp90 ATPase activity with an IC₅₀ of 0.7μM. All these results shed light on the LbHsp90 activity and are the first step to understanding the Hsp90 molecular chaperone system in L. braziliensis.Display Omitted► We studied the structure-function of the Hsp90 of L. braziliensis (LbHsp90). ► LbHsp90 is an elongated dimer in solution. ► The N-domain and NM-domains constructions of the LbHsp90 were monomers. ► The LbHsp90 affinity for adenosine nucleotides and geldanamycin were determined. ► ATPase activity depends on LbHsp90 as dimer and was inhibited by geldanamycin.

Keywords: Abbreviations; AUC; analytical ultracentrifugation; CD; circular dichroism; ƒ/ƒ; ₀; frictional ratio; MM; molecular mass; R; _s; Stokes radius; R; ₀; Stokes radius for a smooth and spherical particle; s; sedimentation coefficient; s; 20,; _w; sedimentation coefficient at standard conditions; s; ⁰; 20,; _w; standard sedimentation coefficient at 0; mg/mL of protein; s; ₀; maximum sedimentation coefficient for a smooth and spherical particle; [θ]; residual molar ellipticity; <; λ; >; center of spectral massHsp90; Molecular chaperone; L. braziliensis; Protozoa

Integral role of the I′-helix in the function of the “inactive” C-terminal domain of catalase–peroxidase (KatG) by Yu Wang; Douglas C. Goodwin (pp. 362-371).

Catalase–peroxidases (KatGs) have two peroxidase-like domains. The N-terminal domain contains the heme-dependent, bifunctional active site. Though the C-terminal domain lacks the ability to bind heme or directly catalyze any reaction, it has been proposed to serve as a platform to direct the folding of the N-terminal domain. Toward such a purpose, its I′-helix is highly conserved and appears at the interface between the two domains. Single and multiple substitution variants targeting highly conserved residues of the I′-helix were generated for intact KatG as well as the stand-alone C-terminal domain (KatG^C). Single variants of intact KatG produced only subtle variations in spectroscopic and catalytic properties of the enzyme. However, the double and quadruple variants showed substantial increases in hexa-coordinate low-spin heme and diminished enzyme activity, similar to that observed for the N-terminal domain on its own (KatG^N). The analogous variants of KatG^C showed a much more profound loss of function as evaluated by their ability to return KatG^N to its active conformation. All of the single variants showed a substantial decrease in the rate and extent of KatG^N reactivation, but with two substitutions, KatG^C completely lost its capacity for the reactivation of KatG^N. These results suggest that the I′-helix is central to direct structural adjustments in the adjacent N-terminal domain and supports the hypothesis that the C-terminal domain serves as a platform to direct N-terminal domain conformation and bifunctionality.Display Omitted► The C-terminal domain may use its I′-helix to adjust N-terminal domain structure. ► I′-helix variants of intact KatG had increasingly reduced stability and activity. ► Multiple substitutions gave properties of the stand-alone N-terminal domain (KatG^N). ► I′-helix variants of KatG^C had diminished capacity to reactivate KatG^N. ► KatG^N reactivation by KatG^C is valuable for exploring KatG structure and function.

Keywords: Abbreviations; KatG; catalase–peroxidase; wtKatG; wild-type KatG; KatG; ^N; separately expressed and isolated KatG N-terminal domain; KatG; ^C; separately expressed and isolated KatG C-terminal domain; LL1; Large Loop 1; LL2; Large Loop 2; APx; ascorbate peroxidase; CcP; cytochrome; c; peroxidase; MCD; magnetic circular dichroism; RHS; rhombic high-spin species; ABTS; 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate); IPTG; isopropyl ß-; d; -thiogalacto-pyranoside; Ni-NTA; nickel nitrilotriacetate; PMSF; phenylmethanesulfonyl fluoride; GuHCl; guanidine hydrochlorideCatalase; Peroxidase; Folding platform; KatG; Gene duplication

Characterization of reaction conditions providing rapid and specific cysteine alkylation for peptide-based mass spectrometry by Jana Paulech; Nestor Solis; Stuart J. Cordwell (pp. 372-379).

Alkylation converts Cys thiols to thioethers and prevents unwanted side reactions, thus facilitating mass spectrometric identification of Cys-containing peptides. Alkylation occurs preferentially at Cys due to its high nucleophilicity, however reactions at other such sites are possible. N-ethylmaleimide (NEM) shows rapid reaction kinetics with Cys and careful definition of reaction conditions results in little reactivity at other sites. Analysis of a protein standard alkylated under differing reaction conditions (pH, NEM concentrations and reaction times) was performed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and selected reaction monitoring (SRM) of NEM-modified and unmodified peptide pairs. Mis-alkylation sites at primary and secondary amines were identified and limited to one equivalent of NEM. No evidence for hydroxyl or thioether alkylation was observed. Improved specificity was achieved by restricting the pH below neutral, NEM concentration below 10mM and/or reaction time to below 5min. Maximal removal of Cys activity was observed in tissue homogenates at 40mM NEM within 1min, dependent upon efficient protein denaturation. SRM assays identified peptide-specific levels of mis-alkylation, indicating that NEM-modified to unmodified ratios did not exceed 10%, with the exception of Cys alkylation that proceeded to 100%, and some Lys residues that resulted in tryptic missed cleavages. High reactivity was observed for His residues considering their relatively low abundance. These data indicate that rapid and specific Cys alkylation is possible with NEM under relatively mild conditions, with more abrasive conditions leading to increased non-specific alkylation without appreciable benefit for MS-based proteomics.► Optimized N-ethylmaleimide Cys alkylation based on concentration, pH and time. ► Near-to-complete Cys alkylation in the absence of non-specific alkylation. ► Improved Cys-peptide recovery in mass spectrometry-based proteomics. ► Cys alkylation prevents artificial Cys modification and improves MS ionization.

Keywords: Abbreviations; NEM; N; -ethylmaleimide; LC-MS/MS; liquid chromatography tandem mass spectrometry; SRM; selected reaction monitoring; IAM; iodoacetamide; DTDP; 4,4′-dithiodipyridine; DTT; dithiothreitolAlkylation; Disulfide; Iodoacetamide; Mass spectrometry; N; -ethylmaleimide; Reduction

A novel domain arrangement in a monomeric cyclodextrin-hydrolyzing enzyme from the hyperthermophile Pyrococcus furiosus by Jong-Tae Park; Hyung-Nam Song; Tae-Yang Jung; Myoung-Hee Lee; Sung-Goo Park; Eui-Jeon Woo; Kwan-Hwa Park (pp. 380-386).

PFTA ( Pyrococcus furiosus thermostable amylase) is a hyperthermophilic amylase isolated from the archaeon Pyrococcus furiosus. This enzyme possesses characteristics of both α-amylase- and cyclodextrin (CD)-hydrolyzing enzymes, allowing it to degrade pullulan, CD and acarbose—activities that are absent in most α-amylases—without the transferring activity that is common in CD-hydrolyzing enzymes. The crystal structure of PFTA revealed a unique monomeric subunit with an extended N-terminal region and an N′-domain folded into its own active site—a significantly altered domain configuration relative to that of the conventional dimeric CD-hydrolyzing amylases in glycoside hydrolase family 13. The active site is formed by the interface of the N′-domain and the catalytic domain and exhibits a broad and wide-open geometry without the concave pocket that is commonly found in the active sites of maltogenic amylases. The mutation of a residue (Gly415 to Glu) located at the domain interface between the N′- and catalytic domains yielded an enzyme that produced a significantly higher purity maltoheptaose (G7) from β-CD, supporting the involvement of this interface in substrate recognition and indicating that this mutant enzyme is a suitable candidate for the production of pure G7. The unique configuration of the active site distinguishes this archaic monomeric enzyme from classical bacterial CD-hydrolyzing amylases and provides a molecular basis for its enzymatic characteristics and for its potential use in industrial applications.Display Omitted► PFTA is a unique hyperthermophilic amylase with cyclodextrin hydrolysis activity. ► The crystal structure of PFTA shows that the enzyme is a self-sufficient monomer. ► The extended N-terminal region folded into its own active site. ► Mutant at the domain interface produced higher purity of maltoheptaose. ► Result supports functional substitution of the N′‐domain in hyperthermophilic archaea.

Keywords: Hyperthermophilic enzyme; Monomer; Substrate specificity; Maltoheptaose production; GH13 family

A ternary complex consisting of AICD, FE65, and TIP60 down-regulates Stathmin1 by Muller Thorsten Müller; Schrotter Andreas Schrötter; Christina Loosse; Kathy Pfeiffer; Carsten Theiss; Marion Kauth; Helmut E. Meyer; Katrin Marcus (pp. 387-394).

The ternary complex consisting of AICD/FE65/TIP60 is thought to play a role in gene expression and was suggested to have a crucial impact in Alzheimer's disease. AICD is the intracellular subdomain of the amyloid precursor protein (APP) and able to bind the adapter protein FE65 and the histone acetyltransferase TIP60 setting up a nuclear dot-like phenotype. Within this work we readdressed the generation of the complex as a function of its compartments. Subsequently, we studied the proteome of AFT expressing cells vs. controls and identified Stathmin1 significantly down-regulated in AFT cells. Stathmin1 functions as an important regulatory protein of microtubule dynamics and was found associated with neurofibrillary tangles in brains of Alzheimer's disease patients. We validated our results using an independent label-free mass spectrometry based method using the same cell culture model. In a reversal model with diminished APP expression, caused by simultaneous knock-down of all three members of the APP family, we further confirmed our results, as Stathmin1 was regulated in an opposite fashion. We hypothesize that AICD-dependent deregulation of Stathmin1 causes microtubule disorganization, which might play an important role for the pathophysiology of Alzheimer's disease.► We studied the proteome of AICD–FE65–TIP60 (AFT) expressing cells vs. controls. ► We identified a new candidate protein significantly down-regulated in AFT cells. ► We confirmed our findings in an independent knock-down model. ► We confirm that AFT establishes a nuclear dot-like phenotype. ► We demonstrate that wtAPP and swAPP are able to generate the AFT complex as well.

Keywords: Alzheimer's disease; APP processing; APP intracellular domain; AICD–FE65 complex; Microtubule disorganization

Barley γ3-hordein: Glycosylation at an atypical site, disulfide bridge analysis, and reactivity with IgE from patients allergic to wheat by Snegaroff Jacques Snégaroff; Isabelle Bouchez; Mohamed El Amine Smaali; Catherine Pecquet; Nadia Raison-Peyron; Pascale Jolivet; Lauriere Michel Laurière (pp. 395-403).

Post translational modifications of a seed storage protein, barley γ3-hordein, were determined using immunochemical and mass spectrometry methods. IgE reactivity towards this protein was measured using sera from patients diagnosed with allergies to wheat. N-glycosylation was found at an atypical Asn-Leu-Cys site. The observed glycan contains xylose. This indicates that at least some γ3-hordein molecules trafficked through the Golgi apparatus. Disulfide bridges in native γ3-hordein were almost the same as those found in wheat γ46-gliadin, except the bridge involving the cysteine included in the glycosylation site. IgE reacted more strongly towards the recombinant than the natural γ3-hordein protein. IgE binding to γ3-hordein increased when the protein sample was reduced. Glycosylation and disulfide bridges therefore decrease epitope accessibility. Thus the IgE from patients sensitized to wheat cross-react with γ3-hordein due to sequence homology with wheat allergens rather than through shared carbohydrate determinants.► The γ3 hordein, a barley prolamin, is glycosylated at an atypical Asn-Leu-Cys site. ► Glycosylated hordein molecules are targeted through the Golgi apparatus. ► Native γ3 hordein disulfide bridge folding is similar to that of wheat γ46-gliadin. ► γ3 hordein binds IgE from wheat-allergic patients mainly after reduction–alkylation. ► γ3 hordein glycosylation is not involved in IgE binding and even decreases it.

Keywords: Abbreviations; CAM; carbamidomethylated; DIG; digoxigenin; IHHWP; immediate hypersensitivity to hydrolyzed wheat protein; LMW-GS; low molecular weight glutenin subunit; TFMS; trifluoromethanesulfonic acid; WDEIA; wheat-dependent exercise-induced anaphylaxisDisulfide bridge; Glycosylation; IgE reactivity; Mass spectrometry; Prolamin; Wheat allergy

Understanding the importance of the aromatic amino-acid residues as hot-spots by I.S. Moreira; J.M. Martins; R.M. Ramos; P.A. Fernandes; M.J. Ramos (pp. 404-414).

Protein–protein interactions (PPI) are crucial for the establishment of life. However, its basic principles are still elusive and the recognition process is yet to be understood. It is important to look at the biomolecular structural space as a whole, in order to understand the principles behind conformation–function relationships. Since the application of an alanine scanning mutagenesis (ASM) study to the growth hormone it was demonstrated that only a small subset of residues at a protein–protein interface is essential for binding — the hot-spots (HS). Aromatic residues are some of the most typical HS at a protein–protein interface. To investigate the structural role of the interfacial aromatic residues in protein–protein interactions, we performed Molecular Dynamic (MD) simulations of protein–protein complexes in a water environment and calculated a variety of physical–chemical characteristics. ASM studies of single residues and of dimers or high-order clusters were performed to check for cooperativity within aromatic residues. Major differences were found between the behavior of non-HS aromatic residues and HS aromatic residues that can be used to design drugs to block the critical interactions or to predict major interactions at protein–protein complexes.► We performed computational measurements to study aromatic interactions. ► vdW contribution is the determinant energetic term. ► Aromatic hot-spots were shown to be protected from bulk water. ► Aromatic residues tend to form dimers or high-order clusters.

Keywords: Alanine scanning mutagenesis; Hot-spot; Aromatic residue; Aromatic cluster; Cooperativity; SASA

Chemistry and mechanism of phosphatases, diesterases and triesterases by Alvan C. Hengge (pp. 415-416).

Efficient, crosswise catalytic promiscuity among enzymes that catalyze phosphoryl transfer by Mark F. Mohamed; Florian Hollfelder (pp. 417-424).

The observation that one enzyme can accelerate several chemically distinct reactions was at one time surprising because the enormous efficiency of catalysis was often seen as inextricably linked to specialization for one reaction. Originally underreported, and considered a quirk rather than a fundamental property, enzyme promiscuity is now understood to be important as a springboard for adaptive evolution. Owing to the large number of promiscuous enzymes that have been identified over the last decade, and the increased appreciation for promiscuity's evolutionary importance, the focus of research has shifted to developing a better understanding of the mechanistic basis for promiscuity and the origins of tolerant or restrictive specificity. We review the evidence for widespread crosswise promiscuity amongst enzymes that catalyze phosphoryl transfer, including several members of the alkaline phosphatase superfamily, where large rate accelerations between 10⁶ and 10¹⁷ are observed for both native and multiple promiscuous reactions. This article is part of a Special Issue entitled: Chemistry and mechanism of phosphatases, diesterases and triesterases.

Keywords: Phosphatase; Catalytic promiscuity; Enzyme mechanism; Sulfate transfer; Sulfatase; Linear-free energy relationship

Promiscuity comes at a price: Catalytic versatility vs efficiency in different metal ion derivatives of the potential bioremediator GpdQ by Lena J. Daumann; Bianca Y. McCarthy; Kieran S. Hadler; Tracy P. Murray; Lawrence R. Gahan; James A. Larrabee; David L. Ollis; Gerhard Schenk (pp. 425-432).

The glycerophosphodiesterase from Enterobacter aerogenes (GpdQ) is a highly promiscuous dinuclear metallohydrolase with respect to both substrate specificity and metal ion composition. While this promiscuity may adversely affect the enzyme's catalytic efficiency its ability to hydrolyse some organophosphates (OPs) and by-products of OP degradation have turned GpdQ into a promising candidate for bioremedial applications. Here, we investigated both metal ion binding and the effect of the metal ion composition on catalysis. The prevalent in vivo metal ion composition for GpdQ is proposed to be of the type Fe(II)Zn(II), a reflection of natural abundance rather than catalytic optimisation. The Fe(II) appears to have lower binding affinity than other divalent metal ions, and the catalytic efficiency of this mixed metal center is considerably smaller than that of Mn(II), Co(II) or Cd(II)-containing derivatives of GpdQ. Interestingly, metal ion replacements do not only affect catalytic efficiency but also the optimal pH range for the reaction, suggesting that different metal ion combinations may employ different mechanistic strategies. These metal ion-triggered modulations are likely to be mediated via an extensive hydrogen bond network that links the two metal ion binding sites via residues in the substrate binding pocket. The observed functional diversity may be the cause for the modest catalytic efficiency of wild-type GpdQ but may also be essential to enable the enzyme to evolve rapidly to alter substrate specificity and enhance k_cat values, as has recently been demonstrated in a directed evolution experiment. This article is part of a Special Issue entitled: Chemistry and mechanism of phosphatases, diesterases and triesterases.► GpdQ is a highly promiscuous phosphoesterase and is useful as a bioremediator. ► The metal ion composition modulates the reaction mechanism. ► The catalytic efficiency of GpdQ is modest. ► Modest catalytic efficiency may be the basis for rapid functional adaptability.

Keywords: Binuclear metallohydrolases; Bioremediation; Glycerophosphodiesterase; Metal ion replacement; Reaction mechanism

Development of metal-ion containing catalysts for the decomposition of phosphorothioate esters by David R. Edwards; R. Stan Brown (pp. 433-442).

The widespread use of phosphorothioate esters as agricultural pesticides, chemical weapons and mechanistic probes in enzymology has sparked interest in the reactivity of these thio-substituted analogues of phosphate esters. In this brief account, we summarize the recent developments in our understanding of the mechanisms of hydrolysis (and solvolysis in methanol) of phosphorothioates containing a sulfur atom in the bridging and/or non-bridging position. A small number of highly efficient catalytic systems containing the metal ions La(III), Pd(II), Cu(II) and Zn(II) have been developed to promote the degradation of the various classes of phosphorothioate esters. The mechanisms of the base promoted solvolytic reactions in water and methanol and those of the metal catalyzed cleavage are presented, as well as a discussion of the energetics of the catalytic processes and other salient features. The aim of this review is to provide the reader with a contemporary physical organic description of phosphorothioate ester cleavage. This article is part of a Special Issue entitled: Chemistry and mechanism of phosphatases, diesterases and triesterases.► Discuss categories of phosphorothioate (PPT) esters and reactions in solution. ► M^x+ catalysts promote the cleavage of PPT triesters with a bridging S in methanol. ► Discussion of the Pd(II) catalysts promoting the solvolysis of PPT pesticides. ► Quantitative measure of Pd(II) catalysis for a methanolysis of a PPT pesticide. ► Transition state bound by catalyst strong enough to convert it into intermediate.

Keywords: Phosphorothioate; Phosphate; Triester; Diester; Hydrolysis; Methanolysis

Catalytic mechanisms for phosphotriesterases by Andrew N. Bigley; Frank M. Raushel (pp. 443-453).

Phosphotriesters are one class of highly toxic synthetic compounds known as organophosphates. Wide spread usage of organophosphates as insecticides as well as nerve agents has lead to numerous efforts to identify enzymes capable of detoxifying them. A wide array of enzymes has been found to have phosphotriesterase activity including phosphotriesterase (PTE), methyl parathion hydrolase (MPH), organophosphorus acid anhydrolase (OPAA), diisopropylfluorophosphatase (DFP), and paraoxonase 1 (PON1). These enzymes differ widely in protein sequence and three-dimensional structure, as well as in catalytic mechanism, but they also share several common features. All of the enzymes identified as phosphotriesterases are metal-dependent hydrolases that contain a hydrophobic active site with three discrete binding pockets to accommodate the substrate ester groups. Activation of the substrate phosphorus center is achieved by a direct interaction between the phosphoryl oxygen and a divalent metal in the active site. The mechanistic details of the hydrolytic reaction differ among the various enzymes with both direct attack of a hydroxide as well as covalent catalysis being found. This article is part of a Special Issue entitled: Chemistry and mechanism of phosphatases, diesterases and triesterases.► The role of binuclear metal clusters in phosphotriester hydrolysis. ► The mechanism of action for the hydrolysis of oganophosphate triesters. ► Variations in protein structure for various phosphotriesterases.

Keywords: Phosphotriesterase; Reaction mechanism

New light on phosphate transfer from triesters by Anthony J. Kirby; José R. Mora; Faruk Nome (pp. 454-463).

The reactivity of triesters is discussed in the general context of phosphate transfer, as usually studied for the reactions of mono- and diesters. Systematic work has typically concentrated on the Linear Free Energy Relationships measuring the dependence of reactivity on the nucleophile and the leaving group, but new results indicate that it can depend equally strongly on the two non-leaving (sometimes known as spectator) groups. This conclusion is supported by first results from theoretical calculations: which also predict that a two-step mechanism can be favored over a concerted S_N2(P) mechanism even for reactions involving leaving groups as good as p-nitrophenolate. This article is part of a Special Issue entitled: Chemistry and mechanism of phosphatases, diesterases and triesterases.Intrinsic Reaction Coordinate for the hydrolysis of DPPNO2P in the presence of 3 water molecules. Structures of the transition states and intermediate are shown.Display Omitted► Changing views and insights on phosphate transfer. ► Important effect of non-leaving groups in hydrolysis of phosphate triesters. ► Theoretical calculations favor two step mechanism in phosphate triester hydrolysis.

Keywords: Phosphate transfer; LFER; Hydrolysis; Density Functional Theory

Redox regulation of protein tyrosine phosphatase activity by hydroxyl radical by Fan-Guo Meng; Zhong-Yin Zhang (pp. 464-469).

Substantial evidence suggests that transient production of reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂) is an important signaling event triggered by the activation of various cell surface receptors. Major targets of H₂O₂ include protein tyrosine phosphatases (PTPs). Oxidation of the active site Cys by H₂O₂ abrogates PTP catalytic activity, thereby potentially furnishing a mechanism to ensure optimal tyrosine phosphorylation in response to a variety of physiological stimuli. Unfortunately, H₂O₂ is poorly reactive in chemical terms and the second order rate constants for the H₂O₂-mediated PTP inactivation are ~10M−¹s−¹, which is too slow to be compatible with the transient signaling events occurring at the physiological concentrations of H₂O₂. We find that hydroxyl radical is produced from H₂O₂ solutions in the absence of metal chelating agent by the Fenton reaction. We show that the hydroxyl radical is capable of inactivating the PTPs and the inactivation is active site directed, through oxidation of the catalytic Cys to sulfenic acid, which can be reduced by low molecular weight thiols. We also show that hydroxyl radical is a kinetically more efficient oxidant than H₂O₂ for inactivating the PTPs. The second-order rate constants for the hydroxyl radical-mediated PTP inactivation are at least 2–3 orders of magnitude higher than those mediated by H₂O₂ under the same conditions. Thus, hydroxyl radical generated in vivo may serve as a more physiologically relevant oxidizing agent for PTP inactivation. This article is part of a Special Issue entitled: Chemistry and mechanism of phosphatases, diesterases and triesterases.► ROS are implicated as mediators of cell-signaling responses by targeting the PTPs. ► The exact identity of the oxidants that inactivate the PTPs is unknown. ► We show that the hydroxyl radical is capable of inactivating the PTPs. ► We also show that hydroxyl radical is a kinetically more efficient oxidant than H₂O₂. ► Hydroxyl radical is a more physiologically relevant oxidant for PTP inactivation.

Keywords: Protein tyrosine phosphatase; Hydrogen peroxide; Hydroxyl radical; Redox regulation

PN bond protein phosphatases by Paul V. Attwood (pp. 470-478).

The current work briefly reviews what is currently known about protein phosphorylation on arginine, lysine and histidine residues, where PN bonds are formed, and the protein kinases that catalyze these reactions. Relatively little is understood about protein arginine and lysine kinases and the role of phosphorylation of these residues in cellular systems. Protein histidine phosphorylation and the two-component histidine kinases play important roles in cellular signaling systems in bacteria, plants and fungi. Their roles in vertebrates are much less well researched and there are no protein kinases similar to the two-component histidine kinases. The main focus of the review however, is to present current knowledge of the characterization, mechanisms of action and biological roles of the phosphatases that catalyze the hydrolysis of these phosphoamino acids. Very little is known about protein phosphoarginine and phospholysine phosphatases, although their existence is well documented. Some of these phosphatases exhibit very broad specificity in terms of which phosphoamino acids are substrates, however there appear to be one or two quite specific protein phospholysine and phosphoarginine phosphatases. Similarly, there are phosphatases with broad substrate specificities that catalyze the hydrolysis of phosphohistidine in protein substrates, including the serine/threonine phosphatases 1, 2A and 2C. However there are two, more specific, protein phosphohistidine phosphatases that have been well characterized and for which structures are available, SixA is a phosphatase associated with two-component histidine kinase signaling in bacteria, and the other is found in a number of organisms, including mammals. This article is part of a Special Issue entitled: Chemistry and mechanism of phosphatases, diesterases and triesterases.► The roles of protein phosphorylation on basic amino acids are not well understood. ► A number of phosphatases have broad specificities for the basic phosphoamino acids. ► SixA is a histidine phosphatase in bacterial two-component histidine kinase systems. ► PHP is a mammalian histidine phosphatase involved with T-cell activation, metastasis.

Keywords: Abbreviations; NDPK; nucleoside diphosphate kinase; PP; protein phosphatase; ACL; ATP-citrate lyase; PHP; protein phosphohistidine phosphataseHistidine kinase; Arginine kinase; Lysine kinase; Phosphohistidine phosphatase; Phosphoarginine phosphatase; Phospholysine phosphatase

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BBA - Proteins and Proteomics (v.1834, #1)