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BBA - Proteins and Proteomics (v.1764, #1)

Editorial Board (pp. ii).

Acknowledgment (pp. 1-4).

NMR mapping of copper binding sites in alpha-synuclein by Yoon-hui Sung; Carla Rospigliosi; David Eliezer (pp. 5-12).

Copper binding to the Parkinson disease-linked protein alpha-synuclein (aS) has been shown to accelerate its oligomerization in vitro and may therefore play a role in aS-mediated pathology in vivo. We use NMR spectroscopy to identify a number of independent copper binding sites in both the lipid-binding N-terminal domain and the highly acidic C-terminal domain of aS. Most of the sites appear to involve negatively charged amino acid side chains, but binding is also observed to the sole histidine residue located at position 50 and to the N-terminal amino group. Both the N-terminal and the histidine sites, as well as the sites in the C-terminal tail, can also bind copper in the more highly structured conformation adopted by aS upon binding to detergent micelles or lipid vesicles. There is no evidence for the formation of any sites requiring long-range order in the protein.

Keywords: Synuclein; Parkinson's disease; Copper binding; Protein aggregation

Biochemical characterization of the class B acid phosphatase (AphA) of Escherichia coli MG1655 by Claudio Passariello; Costantino Forleo; Vanna Micheli; Serena Schippa; Rosalida Leone; Stefano Mangani; Maria Cristina Thaller; Gian Maria Rossolini (pp. 13-19).

The AphA enzyme of Escherichia coli, a molecular class B periplasmic phosphatase that belongs to the DDDD superfamily of phosphohydrolases, was purified and subjected to biochemical characterization. Kinetic analysis with several substrates revealed that the enzyme essentially behaves as a broad-spectrum nucleotidase highly active on 3′- and 5′-mononucleotides and monodeoxynucleotides, but not active on cyclic nucleotides, or nucleotides di- and triphosphate. Mononucleotides are degraded to nucleosides, and AphA apparently does not exhibit any nucleotide phosphomutase activity. However, it can transphosphorylate nucleosides in the presence of phosphate donors. Kinetic properties of AphA are consistent with structural data, and suggest a role for the hydrophobic pocket present in the active site crevice, made by residues Phe 56, Leu71, Trp77 and Tyr193, in conferring preferential substrate specificity by accommodating compounds with aromatic rings. AphA was inhibited by several chelating agents, including EDTA, EGTA, 1,10-phenanthroline and dipicolinic acid, with EDTA being apparently the most powerful inhibitor.

Keywords: AphA; class B acid phosphatase; Escherichia coli; Kinetic analysis; Biochemical characterization

Analysis of interaction between DNA and Deinococcus radiodurans PprA protein by Atomic force microscopy by Masahiro Murakami; Issay Narumi; Katsuya Satoh; Akira Furukawa; Isamu Hayata (pp. 20-23).

A DNA repair-promoting protein, PprA, was isolated from a radiation resistant bacterium, Deinococcus radiodurans [I. Narumi, K. Sato, S. Cui, T. Funayama, S. Kitayama, H. Watanabe, PprA: a novel protein from Deinococcus radiodurans that stimulates DNA ligation, Mol. Microbiol. 54 (2004) 278–285]. Despite several studies, however, the function of PprA is not still clear. We used atomic force microscopy (AFM) to elucidate the role of this protein in the DNA repair pathway. In the present study, interaction between the linear DNA and PprA protein was imaged and analyzed by AFM without any fixation or staining. Though both end-bound and internally bound PprA was observed, the affinity of the end-bound protein was greater considering the proportion of features of binding analyzed by AFM. In some conditions, looping forms of the DNA–PprA complex were observed. Gel filtration high performance liquid chromatography (HPLC) was also conducted to estimate the molecular weight of this protein. The result of the HPLC analysis suggested that PprA formed multimers in buffer solution without DNA.

Keywords: Atomic force microscopy; DNA repair; Radioresistant bacterium; Deinococcus radiodurans

Synergism of Leu–Lys rich antimicrobial peptides and chloramphenicol against bacterial cells by Yoonkyung Park; Soon Nang Park; Seong-Cheol Park; Sun Oh Shin; Jin-Young Kim; Sung-Jin Kang; Mi-Hyun Kim; Chan-Young Jeong; Kyung-Soo Hahm (pp. 24-32).

To investigate the antibiotic activity and synergistic effect, analogues were designed to increase not only net positive charge by Lys-substitution but also hydrophobic helix region by Leu-substitution from CA (1–8)-MA (1–12) hybrid peptide (CA-MA). In particular, CA-MA analogue P5 (P5), designed by flexible region (GIG→P)-substitution, Lys- (positions 4, 8, 14, 15) and Leu- (positions 5, 6, 12, 13, 16, 17, 20) substitutions, showed potent antibacterial activity in minimal inhibition concentration (MIC) and minimal bactericidal concentration (MBC) without having hemolytic activity. In addition, P5 and chloramphenicol has potent synergistic effect against tested cell lines. As determined by propidium iodide (PI) staining, flow cytometry showed that P5 plus chloramphenicol-treated cells had higher fluorescence intensity than untreated, P5- and chloramphenicol-treated cells. The effect on plasma membrane was examined by investigating the transmembrane potential depolarizing experiments of S. aureus with P5 and chloramphenicol. The result showed that the peptide exerts its antibacterial activity by acting on the plasma membrane. Furthermore, P5 caused significant morphological alterations of S. aureus, as shown by scanning electron microscopy. Our results suggest that peptide P5 is an excellent candidate as a lead compound for the development of novel antiinfective agents and synergistic effects with conventional antibiotic agents but lack hemolytic activity.

Keywords: Synergistic effect; Positive charge; CA (1–8)-MA (1–12) hybrid peptide; CA-MA analogue P5; Minimal bactericidal concentration; Scanning electron microscopy

Molecular characterization of a novel dipeptidyl peptidase like 2-short form (DPL2-s) that is highly expressed in the brain and lacks dipeptidyl peptidase activity by Tong Chen; Katerina Ajami; Geoffrey W. McCaughan; Wei-Ping Gai; Mark D. Gorrell; Catherine A. Abbott (pp. 33-43).

DPL2 (DPP10) found at chromosome 2q14.1 is a member of the dipeptidyl peptidase IV (DPIV) gene family. Here we characterize a novel short DPL2 isoform (DPL2-s), a 789-amino acid protein, that differs from the previously described long DPL2 isoform (DPL2-l) at the N-terminal cytoplasmic domain by 13 amino acids. The two DPL2 isoforms use alternate first exons. DPL2 mRNA was expressed mainly in the brain and pancreas. Multiple forms of recombinant DPL2-s protein were observed in 293T cells, having mobilities 96 kDa, 100 kDa, and approximately 250 kDa which may represent soluble DPL2, transmembrane DPL2 and multimeric DPL2 respectively. DPL2 is glycosylated as a band shift is observed following PNGase F deglycosylation. DPL2-s was expressed primarily on the cell surface of transfected 293T and PC12 cells. DPL2-s exhibits high sequence homology with other DPIV peptidases, but lacks a catalytic serine residue and lacks dipeptidyl peptidase activity. Substitutions of Gly⁶⁴⁴→Ser, Lys⁶⁴³Gly⁶⁴⁴→TrpSer, or Asp⁵⁶¹Lys⁶⁴³Gly⁶⁴⁴→TyrTrpSer in the catalytic motif did not confer dipeptidyl peptidase activity upon DPL2-s. Thus, although DPL2 is similar in structure and sequence to the other dipeptidyl peptidases, it lacks vital residues required to confer dipeptidyl peptidase activity and has instead evolved features that enable it to act as an important component of voltage-gated potassium channels.

Keywords: Abbreviations; BLAST; Basic Local Alignment Search Tool; DP; dipeptidyl peptidase; DPL; DP-like; FAP; fibroblast activation protein; I; _SA; A-type Kv current; Kv channel; voltage-gated potassium channel; KChIP; Kv channel interacting protein; GFP; green fluorescent protein; NGF; nerve growth factorDPL2; DPIV; DPL1; Kv channel; Serine protease

Three-dimensional domain-swapped oligomers of ribonuclease A: identification of a fifth tetramer, pentamers and hexamers, and detection of trace heptameric, octameric and nonameric species by Giovanni Gotte; Douglas V. Laurents; Massimo Libonati (pp. 44-54).

By lyophilization from 40% acetic acid solutions, bovine pancreatic Ribonuclease A forms three-dimensional domain-swapped dimers, trimers, and tetramers that can be separated by cation-exchange chromatography. Each oligomeric species consists of at least two conformers, one less basic, one more basic. The structures of the two dimers and one trimer have been solved. Plausible models have been proposed for a second RNase A trimer and four tetramers. This work is focused on the characterization of the largest oligomers which compose small peaks that have always appeared in chromatograms of RNase A. These higher order oligomers were collected by repeated cation-exchange chromatographies. On the basis of (a) gel filtrations through analytical Superdex 75 and 200; (b) gel electrophoreses under non-denaturing conditions, (c) cross-linking with divynilsulfone followed by analyses with SDS-PAGE and mass spectrometry, (d) enzymatic activity assays, and (e) analyses of the products of spontaneous dissociation of the oligomers, we could identify three-dimensional domain-swapped pentamers and hexamers, and one additional tetrameric conformer. For the latter we propose a cyclic model (C_TT). Moreover, we advance a linear model (NCNC_P) for one pentamer, and three possible cyclic models (with a C-trimer as the main component) for one hexamer. The experimental evidence also indicates the existence of heptameric, octameric and nonameric species.

Keywords: Abbreviations; RNase A; ribonuclease A; poly(A).poly(U), double-stranded; associated polyadenylate and polyuridylate; DVS; divinylsulfoneRNase A oligomer; Three-dimensional domain-swapping; RNase A tetramer; RNase A pentamer; RNase A hexamer

Local protein flexibility as a prerequisite for reversible chromophore isomerization in α-phycoerythrocyanin by Marius Schmidt; Angela Krasselt; Wolfgang Reuter (pp. 55-62).

Phycoerythrocyanin is the only cyanobacterial phycobiliprotein containing phycoviolobilin as a chromophore. The phycoviolobilin chromophore is photo-reactive; upon irradiation, the chromophore undergoes a Z/E-isomerization involving the rotation of pyrrole-ring D. We have determined the structure of trimeric phycoerythrocyanin at three different experimental settings: monochromatically at 110 K and 295 K as well as with the Laue method at 288 K. Based on their chemical structures, the restraints for the phycoviolobilin of the α-subunit and for the phycocyanobilin chromophores of the β-subunit were newly generated, which allows a chemically meaningful refinement of both chromophores. All three phycoerythrocyanin structures are very similar; the subunits match within 0.5 Å. The detailed comparison of the data obtained with the different measurements provided information about the protein properties around the phycoviolobilin chromophore. For the first time, crystals of a phycobilisome protein are used successfully with the Laue technique. This paves the way for time-resolved macromolecular crystallography, which is able to elucidate the exact mechanisms of the phycoviolobilin photoactivity including the protein involvement.

Keywords: Laue crystallography; Phycobiliprotein; Phycoerythrocyanin; Phycocyanin; Z/E; isomerization; Photo-activity

Crystal structure of CTP:glycerol-3-phosphate cytidylyltransferase from Staphylococcus aureus: Examination of structural basis for kinetic mechanism by Desiree H. Fong; Veronica C.-N. Yim; Michael A. D'Elia; Eric D. Brown; Albert M. Berghuis (pp. 63-69).

Integrity of the cell wall is essential for bacterial survival, and as a consequence components involved in its biosynthesis can potentially be exploited as targets for antibiotics. One such potential target is CTP:glycerol-3-phosphate cytidylyltransferase. This enzyme (TarD_Sa in Staphylococcus aureus and TagD_Bs in Bacillus subtilis) catalyzes the formation of CDP-glycerol, which is used for the assembly of linkages between peptidoglycan and teichoic acid polymer in Gram-positive bacteria. Intriguingly, despite the high sequence identity between TarD_Sa and TagD_Bs (69% identity), kinetic studies show that these two enzymes differ markedly in their kinetic mechanism and activity. To examine the basis for the disparate enzymological properties, we have determined the crystal structure of TarD_Sa in the apo state to 3 � resolution, and performed equilibrium sedimentation analysis. Comparison of the structure with that of CTP- and CDP-glycerol-bound TagD_Bs crystal structures reveals that the overall structure of TarD_Sa is essentially the same as that of TagD_Bs, except in the C-terminus, where it forms a helix in TagD_Bs but is disordered in the apo TarD_Sa structure. In addition, TarD_Sa can exist both as a tetramer and as a dimer, unlike TagD_Bs, which is a dimer. These observations shed light on the structural basis for the differing kinetic characteristics between TarD_Sa and TagD_Bs.

Keywords: Cytidylyltransferase; Teichoic acid; CTP; Enzyme mechanism; Protein structure

The study of solution conformation of allatostatins by 2-D NMR and molecular modeling by Zhen-peng Kai; Yun Ling; Wen-jun Liu; Fei Zhao; Xin-ling Yang (pp. 70-75).

More than 70 allatostatins have been isolated from various insects and there is interest in the determination of their active conformation. We have synthesized Dippu-AST 1 (originally isolated from the cockroach Blattella germanica) and studied its conformation in solution by 2-D NMR and molecular modeling. Dippu-AST 1 belongs to the cockroach-type ASTs that have Y/FXFGL-NH₂ as the common C-terminal sequence. We found that Dippu-AST 1 forms a type I' β-turn conformation in DMSO. We also studied the conformations of Dippu-AST 1 and six cockroach-type allatostatins in water using the molecular dynamics method. When the X amino acid in the consensus sequence Y/FXFGL-NH₂ is Ala or Ser, the allatostatin can form a typical type II β-turn. If the X is Asp or Asn whose side chain contains a carbonyl, the allatostatin can form a type I, I' or IV β-turn conformation; if the X is Gly, a closer γ-turn is adopted. Our study indicates that the turn conformation is ubiquitous in cockroach-type allatostatins.

Keywords: Allatostatin; Conformation; 2-D NMR; Molecular dynamics

Proteomic analysis of the venom and characterization of toxins specific for Na⁺- and K⁺-channels from the Colombian scorpion Tityus pachyurus by Jacqueline Barona; Cesar V.F. Batista; Fernando Z. Zamudio; Froylan Gomez-Lagunas; Enzo Wanke; Rafael Otero; Lourival D. Possani (pp. 76-84).

The Colombian scorpion Tityus pachyurus is toxic to humans and is capable of producing fatal accidents, but nothing is known about its venom components. This communication reports the separation of at least 57 fractions from the venom by high performance liquid chromatography. From these, at least 104 distinct molecular weight compounds were identified by mass spectrometry analysis. The complete amino acid sequences of three peptides were determined and the partial sequences of three others were also identified. Electrophysiological experiments conducted with ion-channels expressed heterologously on Sf9 cells showed the presence of a potent Shaker B K⁺-channel blocker. This peptide (trivial name Tpa1) contains 23 amino acid residues closely packed by three disulfide bridges with a molecular mass of 2457 atomic mass units. It is the third member of the sub-family 13, for which the systematic name is proposed to be α-KTx13.3. The mice assay showed clearly the presence of toxic peptides to mammals. One of them named Tpa2, containing 65 amino acid residues with molecular mass of 7522.5 atomic mass units, is stabilized by four disulfide bridges. It was shown to modify the Na⁺-currents of F-11 and TE671 cells in culture, similar to the beta scorpion toxins. These results demonstrate the presence of toxic peptides in the venom of T. pachyurus and confirm that accidents with this species of scorpion should be considered an important human hazard in Colombia.

Keywords: Amino acid sequence; Ion-channel; Mass spectrometry; Proteomic analysis; Scorpion toxin; Tityus pachyurus

Cloning and expression of IspDF from Mesorhizobium loti. Characterization of a bifunctional protein that catalyzes non-consecutive steps in the methylerythritol phosphate pathway by Charles A. Testa; Christian Lherbet; Florence Pojer; Joseph P. Noel; C. Dale Poulter (pp. 85-96).

Gram-negative bacteria, plant chloroplasts, green algae and some Gram-positive bacteria utilize the 2- C-methyl-d-erythritol phosphate (MEP) pathway for the biosynthesis of isoprenoids. IspD, ispE, and ispF encode the enzymes required to convert MEP to 2- C-methyl-d-erythritol 2,4-cyclodiphosphate (cMEDP) during the biosynthesis of isopentenyl diphosphate and dimethylallyl diphosphate in the MEP pathway. Upon analysis of the Mesorhizobium loti genome, ORF mll0395 showed homology to both ispD and ispF and appeared to encode a fusion protein. M. loti ispE was located elsewhere on the chromosome. Purified recombinant IspDF protein was mostly a homodimer, MW ∼46 kDa/subunit. Incubation of IspDF with MEP, CTP, and ATP gave 4-diphosphocytidyl-2- C-methyl-d-erythritol (CDP-ME) as the only product. When Escherichia coli IspE protein was added to the incubation mixture, cMEDP was formed. In addition, M. loti ORF mll0395 complements lethal disruptions in both ispD and ispF in Salmonella typhimurium. These results indicate that IspDF is a bifunctional protein, which catalyzes the first and third steps in the conversion of MEP to cMEDP.

Keywords: Abbreviations; Amp; ampicillin; ara; l; -arabinose; Cam; chloramphenicol; CAT; chloramphenicol acetyl transferase; CDP-ME; 4-diphosphocytidyl-2-; C; -methyl-; d; -erythritol; CDP-MEP; 4-diphosphocytidyl-2-; C; -methyl-; d; -erythritol 2-phosphate; cMEDP; 2-; C; -methyl-; d; -erythritol 2,4-cyclodiphosphate; DMAPP; dimethylallyl diphosphate; DXP; 1-deoxy-; d; -xylulose 5-phosphate; HDMAPP; 1-hydroxy-2-methyl-2-buten-4-yl diphosphate; HMG-CoA; 3-hydroxy-3-methylglutaryl CoA; IPP; isopentenyl diphosphate; Kan; kanamycin; ME; 2-; C; -Methyl-; d; -erythritol; MEP; 2-; C; -methyl-; d; -erythritol 4-phosphate; MVA; mevalonate; PP; _i; inorganic pyrophosphateCloning; IspDF; Mesorhizobium loti

Thermostable DNA polymerases can perform translesion synthesis using 8-oxoguanine and tetrahydrofuran-containing DNA templates by Ekaterina A. Belousova; Nadejda I. Rechkunova; Olga I. Lavrik (pp. 97-104).

The translesion synthesis (TLS) capacity of the thermostable DNA polymerases Taq, Tte and Tte-seq utilizing a synthetic abasic site, tetrahydrofuran (THF), and an 8-oxoguanine-containing DNA template was investigated. Measurements with human DNA polymerase beta were used as a “positive control�?. Thermostable DNA polymerases were observed to perform TLS with different specificities on both substrates. With a THF-containing template, dGMP was preferentially inserted by all the DNA polymerases. In the presence of Mn(II) as a cofactor, all the polymerases incorporated dCMP opposite 8-oxoguanine whereas, in the presence of Mg(II) ions, dAMP was incorporated. It was found that none of the thermophilic DNA polymerases utilized dTTP with either an 8-oxoguanine or a THF-containing template. In all cases, DNA duplex containing THF as damage was processed to full length less effectively than DNA duplex containing 8-oxoguanine.

Keywords: Thermophilic DNA polymerase Taq; Tte; Tte-seq; DNA polymerase beta; Translesion DNA Synthesis; DNA Replication and Repair

Purification and characterization of recombinant Caulobacter crescentus Cu,Zn superoxide dismutase by Ivana De Domenico; Amalia Lania; Maria Carmela Bonaccorsi di Patti; Andrea Battistoni; Giovanni Musci; Alessandro Desideri (pp. 105-109).

Recombinant Cu,Zn Superoxide Dismutase from Caulobacter crescentus has been expressed in Escherichia coli and characterized. The corresponding recombinant protein has a molecular weight typical of a homodimeric Cu,ZnSODs and an activity comparable to that of other prokaryotic enzymes. The copper active site is characterized by a peculiar axial geometry as evidenced by its electron paramagnetic resonance spectrum, moreover, the copper atom displays a low accessibility toward external chelating agents indicating a lower solvent accessibility when compared to other prokaryotic enzymes. Investigation of the enzyme thermal stability through differential scanning calorimetry indicates the occurrence of two transitions at low and higher temperature that are found to be due to the apo and holo protein, respectively, confirming that the metals have a crucial role in the stabilization of this class of enzymes.

Keywords: Abbreviations; SOD; superoxide dismutase; DTT; dithiothreitol; EDTA; ethylenediaminetetraacetic acid; EPR; Electron Paramagnetic Resonance; DSC; Differential Scanning CalorimetrySuperoxide dismutase; Caulobacter crescentus; Copper

Crystal structures of the free and sterol-bound forms of β-cinnamomin by Maria Luisa Rodrigues; Margarida Archer; Paulo Martel; Sandra Miranda; M�nica Thomaz; Francisco Javier Enguita; Ricardo P. Baptista; Eduardo Pinho e Melo; Nelson Sousa; Alfredo Cravador; Maria Arm�nia Carrondo (pp. 110-121).

The crystal structure of the elicitin β-cinnamomin (β-CIN) was determined in complex with ergosterol at 1.1 Å resolution. β-CIN/ergosterol complex crystallized in the monoclinic space group P2₁, with unit cell parameters of a=31.0, b=62.8, c=50.0 Å and β=93.4° and two molecules in the asymmetric unit. Ligand extraction with chloroform followed by crystallographic analysis yielded a 1.35 Å structure of β-CIN (P4₃2₁2 space group) where the characteristic elicitin fold was kept. After incubation with cholesterol, a new complex structure was obtained, showing that the protein retains, after the extraction procedure, its ability to complex sterols. The necrotic effect of β-CIN on tobacco was also shown to remain unchanged. Theoretical docking studies of the triterpene lupeol to β-CIN provided an explanation for the apparent inability of β-CIN to bind this ligand, as observed experimentally.

Keywords: Elicitin; Sterol carrier protein; Crystal structure; Phytophthora

Detection and characterization of merohedral twinning in crystals of oxalyl-coenzyme A decarboxylase from Oxalobacter formigenes by Catrine L. Berthold; Harmeet Sidhu; Stefano Ricagno; Nigel G. Richards; Ylva Lindqvist (pp. 122-128).

Oxalyl-coenzyme A decarboxylase is a thiamin diphosphate dependent enzyme active in the catabolism of the highly toxic compound oxalate. The enzyme from Oxalobacter formigenes has been expressed as a recombinant protein in Escherichia coli, purified to homogeneity and crystallized. Two crystal forms were obtained, one showing poor diffraction and the other merohedral twinning. Crystals in the former category belong to the tetragonal space group P4₂2₁2. Data to 4.1 � resolution were collected from these crystals and an incomplete low resolution structure was initially determined by molecular replacement. Crystals in the latter category were obtained by co-crystallizing the protein with coenzyme A, thiamin diphosphate and Mg²⁺-ions. Data to 1.73 � were collected from one of these crystals with apparent point group 622. The crystal was found to be heavily twinned, and a twin ratio of 0.43 was estimated consistently by different established methods. The true space group P3₁21 was deduced, and a molecular replacement solution was obtained using the low resolution structure as template when searching in detwinned data.

Keywords: Oxalyl-coenzyme A decarboxylase; Thiamin diphosphate; Hemihedral twinning; Twin fraction; X-ray crystallography

Unusual effect of salts on the homodimeric structure of NADH oxidase from Thermus thermophilus in acidic pH by Marek Stupák; Gabriel Žoldák; Andrej Musatov; Mathias Sprinzl; Erik Sedlák (pp. 129-137).

The unusual salt-dependent behavior of the homodimeric flavoenzyme NADH oxidase from Thermus thermophilus in acidic pH has been studied using circular dichroism (CD) and sedimentation velocity. The native-like secondary and quaternary structures in acidic low ionic strength conditions were significantly perturbed by the addition of salts. The peptide region of the CD spectra showed a major salt-induced conformational change in the protein secondary structure. Sedimentation velocity experiments showed dissociation of the homodimeric structure of NADH oxidase in the presence of salt (>1 M). The new acidic conformation of the protein was stabilized by high ionic strength as indicated by a salt-induced increase in the melting temperature of the protein, and by a shift in the apparent p K_a values of the conformational transition to a less acidic pH. Distortion of the dominant α-helical signal was expressed as the disappearance of the parallel polarized Moffitt exciton band at 208 nm without an accompanying loss of amplitude of n→ π* electronic transitions at 222 nm. The unusual CD spectra correlated qualitatively with the theoretically calculated CD spectra of short α-helical structures and/or twisted β-sheets. Differences between the experimentally obtained CD spectra and theoretical calculations (AGADIR) of the α-helical content of NADH oxidase indicate a role for non-local interactions in the protein conformation at high ionic strength and low pH. These findings indicate the importance of the homodimeric interface and electrostatic interactions for maintaining the structural integrity of this thermophilic protein.

Keywords: NADH oxidase; Flavoprotein; Molten globule; Dimeric interface; Circular dichroism

Structure and potential energy surface studies on 3₁₀ helices of hen egg white lysozyme and Phaseolus vulgaris arcelin-1 proteins by P. Kolandaivel; P. Selvarengan; K.V. Gunavathy (pp. 138-145).

Density functional theory studies have been performed for 3₁₀-helix oligomers of hen egg white lysozyme and Phaseolus vulgaris Arcelin-1 Proteins. Severe perturbation in the structure has been noted when the fully optimized structural parameters of oligomers are compared with experimental results. The potential energy surfaces have been generated for all the oligomers. It can be found that no change has been observed in the global minimum structure of Tyrosine–Arginine–Glycine (YRG), but each structure of Glycine–Arginine–Tyrosine (GRY) belongs to different positions in the φ– ψ space. It can be concluded that due to the floppiness of the considered peptides, the molecule fluctuate or interconvert easily between different conformations with different dipole moments pointing in different directions. The substitution of Tyrosine at the N-terminal played major role for the helix formation due to the presence of strong main chain hydrogen bond interaction with glycine. The molecular properties, such as stabilization energy, ionization energy, electron affinity, were calculated and interpreted. The simulated amide bands of the oligomers coincide well with experimental frequencies.

Keywords: Density functional theory; Hen egg white lysozyme; Phaseolus vulgaris; arcelin-1; Potential energy surface; 3; ₁₀; -helix

Structural characterization of novel chitin-binding lectins from the genus Artocarpus and their antifungal activity by Melissa B. Trindade; Jos� L.S. Lopes; Andr�a Soares-Costa; Ana Cristina Monteiro-Moreira; Renato A. Moreira; Maria Luiza V. Oliva; Leila M. Beltramini (pp. 146-152).

Two novel chitin-binding lectins from seeds of Artocarpus genus were described in this paper, one from A. integrifolia (jackfruit) and one from A. incisa (breadfruit). They were purified from saline crude extract of seeds using affinity chromatography on chitin column, size-exclusion chromatography and reverse-phase chromatography on the C-18 column. Both are 14 kDa proteins, made up of 3 chains linked by disulfide bonds. The partial amino acid sequences of the two lectins showed they are homologous to each other but not to other plant chitin-binding proteins. Thus, they cannot be classified in any known plant chitin-binding protein family, particularly because of their inter-chain covalent bonds. Their circular dichroism spectra and deconvolution showed a secondary structure content of β-sheet and unordered elements. The lectins were thermally stable until 80 °C and structural changes were observed below pH 6. Both lectins inhibited the growth of Fusarium moniliforme and Saccharomyces cerevisiae, and presented hemagglutination activity against human and rabbit erythrocytes. These lectins were denoted jackin (from jackfruit) and frutackin (from breadfruit).

Keywords: Abbreviations; CD; circular dichroism; DTT; dithiothreitol; EDTA; ethylenediaminotetraacetic acid disodium salt; LC/ESI/MS; liquid chromatography/electronspray ionization/mass spectrometry; GlcNAc; N; -acetyl-glucosamine; PBS; phosphate buffered saline; PDM; potato dextrose medium; SEC; size exclusion chromatography; TFA; trifluoroacetic acid; HPLC; high performance liquid chromatography; UDA; Urtica dioica agglutinin; WGA; Wheat germ agglutininChitin-binding lectin; Lectin from; Artocarpus; Fusarium moniliforme; Saccharomyces cerevisiae; Antifungal activity

Protein preparation, crystallization and preliminary X-ray analysis of imidazolonepropionase from Bacillus subtilis by Yamei Yu; Lanfen Li; Xiaofeng Zheng; Yu-He Liang; Xiao-Dong Su (pp. 153-156).

Imidazolonepropionase (EC 3.5.2.7) is the third enzyme of the histidine degradation pathway that has been conserved from bacteria to eukaryotes. The enzyme is the only one with unknown three-dimensional structure in this pathway. In this work, Bacillus subtilis imidazolonepropionase (HutI) was expressed in E. coli and purified to homogeneity. After thrombin digestion, high quality crystals were obtained by hanging-drop vapor diffusion method. The best crystal diffracted to 2.0 Å and belonged to the space group P2₁ with unit-cell parameters a=57.73 Å, b=106.34 Å, c=66.47 Å, β=89.93 °.

Keywords: Imidazolonepropionase; Crystallization; Thrombin-digestion; X-ray diffraction

Crystallization and preliminary crystallographic analysis of the N-terminal domain of PriA from Escherichia coli by Kaori Sasaki; Toyoyuki Ose; Taku Tanaka; Toshimi Mizukoshi; Tomoko Ishigaki; Katsumi Maenaka; Hisao Masai; Daisuke Kohda (pp. 157-160).

PriA, a DEXH-type DNA helicase, binds specifically to the 3′ end of DNA through its N-terminal domain, and is a candidate sensor protein that recognizes arrested DNA replication forks in bacteria. We crystallized an N-terminal fragment of PriA in the absence and the presence of oligonucleotides to elucidate the structural basis for the specific recognition of the 3′ terminus of DNA.

Keywords: PriA; Escherichia coli; Crystallization; 3′-terminal nucleotide; Arrested replication fork

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BBA - Proteins and Proteomics (v.1764, #1)