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BBA - Proteins and Proteomics (v.1794, #4)

Editorial Board (pp. i).

A biophysical insight into the RANTES–glycosaminoglycan interaction by Angelika Rek; Barbara Brandner; Elena Geretti; Andreas J. Kungl ⁎ (pp. 577-582).

Binding of chemokines to glycosaminoglycans (GAGs) represents a crucial step in leukocyte attraction and activation. Since chemokine oligomerisation was shown to be important for GAG binding, the apparent oligomerisation constant of RANTES was determined to be 225 nM using fluorescence anisotropy. In the presence of heparan sulfate, chemokine oligomerisation was found to be promoted by the glycosaminoglycan as expressed in the increase in cooperativity and a shift towards higher melting temperatures in thermal unfolding experiments. In surface plasmon resonance investigations of RANTES–GAG binding kinetics using a heparan sulfate-coated chip, GAG-induced oligomerisation led to a bell-shaped (bi-phasic) Scatchard plot referring to cooperativity in the chemokine–GAG interaction. This was absent in the oligomerisation-deficient RANTES mutants N46R and Q48K. We have further investigated the dependence of RANTES–GAG dissociation constants on oligosaccharide chain length by performing isothermal fluorescence titrations with size-defined heparin and heparan sulfate oligosaccharides as chemokine ligands. Heparin dp18 and heparan sulfate dp14 yielded the highest affinities with K_d values of 31.7 nM and 42.9 nM, respectively. Far-UV CD spectroscopy revealed a significant conformational change of RANTES upon heparan sulfate binding which is suggested to be a pre-requisite for oligomerisation and thus for optimal GPCR activation in vivo. This was shown by the impaired chemotactic activity of the RANTES N46R and Q48K mutants.

Keywords: Abbreviations; CD; circular dichroism; GAG; glycosaminoglycan; HS; heparan sulfate; RANTES; regulated upon activation normal T cell expressed and secreted; dp; degree of polymerisation; bis-ANS; 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid; PBS; phosphate buffered saline; GdmCl; guanidinium hydrochlorideHeparin; Heparan sulfate; Chemokine; Fluorescence spectroscopy; Circular dichroism

Analysis of conservation in the Fas-associated death domain protein and the importance of conserved tryptophans in structure, stability and folding by Hai Li; Jessica L. Wojtaszek; Lesley H. Greene ⁎ (pp. 583-593).

Computational and experimental studies focusing on the role of conserved amino acids for folding and stability is an active and promising area of research. To date however, only a small fraction of the potential superfamilies have been investigated. To further expand our understanding we present the results of a bioinformatics analysis of the death domain superfamily. The fold consists of a six helical bundle with a Greek-key topology. Our sequence and structural studies have identified a group of conserved hydrophobic residues and corresponding long-range interactions, which we propose are important in the formation and stabilization of the hydrophobic core and native topology. Six conserved hydrophilic residues were additionally identified and may play a functional role during apoptosis. We also report the establishment of an experimental system that will facilitate studies to test the role of the conserved residues in folding and stability. Equilibrium unfolding and refolding studies of a model superfamily member, Fas-associated death domain protein indicate that the process is cooperative, two-state and reversible. Stopped-flow fluorescence studies reveal that the folding is rapid and biphasic with the majority of the hydrophobic core forming in the first phase. Site-directed mutagenesis studies indicate that conserved tryptophans 112 and 148 are important to structure, native state stability and folding. These results present the earliest conservation analysis and biophysical characterization of the Fas-associated death domain.

Keywords: Abbreviations; DD; Death domain; DED; Death effector domain; CARD; Caspase recruitment domain; PYD; Pyrin domain; Fadd-DD; Fas-associated death domain protein; WT; Wild-type; CD; Circular dichroismFas-associated death domain protein; Protein folding; Stability; Conserved residue; Greek-key topology; Death domain superfamily

Studies of interaction of homo-dimeric ferredoxin-NAD(P)⁺ oxidoreductases of Bacillus subtilis and Rhodopseudomonas palustris, that are closely related to thioredoxin reductases in amino acid sequence, with ferredoxins and pyridine nucleotide coenzymes by Daisuke Seo ⁎; Seisuke Okabe; Mitsuhiro Yanase; Kunishige Kataoka; Takeshi Sakurai (pp. 594-601).

Ferredoxin-NADP⁺ oxidoreductases (FNRs) of Bacillus subtilis (YumC) and Rhodopseudomonas palustris CGA009 (RPA3954) belong to a novel homo-dimeric type of FNR with high amino acid sequence homology to NADPH-thioredoxin reductases. These FNRs were purified from expression constructs in Escherichia coli cells, and their steady-state reactions with [2Fe–2S] type ferredoxins (Fds) from spinach and R. palustris, [4Fe–4S] type Fd from B. subtilis, NAD(P)⁺/NAD(P)H and ferricyanide were studied. From the K_m and k_cat values for the diaphorase activity with ferricyanide, it is demonstrated that both FNRs are far more specific for NADPH than for NADH. The UV–visible spectral changes induced by NADP⁺ and B. subtilis Fd indicated that both FNRs form a ternary complex with NADP⁺ and Fd, and that each of the two ligands decreases the affinities of the others. The steady-state kinetics of NADPH-cytochrome c reduction activity of YumC is consistent with formation of a ternary complex of NADPH and Fd during catalysis. These results indicate that despite their low sequence homology to other FNRs, these enzymes possess high FNR activity but with measurable differences in affinity for different types of Fds as compared to other more conventional FNRs.

Keywords: Ferredoxin; Ferredoxin-NADP; ⁺; oxidoreductase; Firmicute; Green sulfur bacterium; FNR

Conformational and biochemical characterization of a biologically active rat recombinant Protease Nexin-1 expressed in E. coli by Rosaria Arcone ⁎; Alberto Chinali; Nicola Pozzi; Maddalena Parafati; Fabio Maset; Concetta Pietropaolo; Vincenzo De Filippis ⁎ (pp. 602-614).

Protease Nexin-1, a 43-kDa glycoprotein, is a major physiological thrombin inhibitor involved in the modulation of nerve cell plasticity. Recombinant rat Protease Nexin-1 (rPN-1) was efficiently produced in Escherichia coli using a T7 RNA polymerase based expression system and purified by heparin-sepharose affinity chromatography yielding 3 mg of protein per liter of cell culture. The purity and chemical identity of rPN-1 were assessed by SDS-PAGE, Reverse Phase- High Performance Liquid Chromatography, mass spectrometry and two-dimensional-gel electrophoresis. Conformational analysis by circular dichroism and fluorescence spectroscopy revealed the presence of mixed α/β secondary structure and the prevailing localization of Trp-residues in rather polar environments. Fluorescence titration of rPN-1 with heparin indicated that rPN-1 binds heparin with high affinity. Furthermore, the formation of a SDS-stable 1:1 thrombin–rPN-1 complex, monitored by SDS-PAGE, confirmed the native-like structure of rPN-1. Finally, the cellular effects of rPN-1, such as its ability to promote neurite outgrowth in neuroblastoma cells, were found to be very similar to those elicited by natural PN-1. Altogether, our results demonstrate that glycosylation does not alter neither structure nor function of PN-1 and that E. coli is a suitable expression system for obtaining milligram quantities of pure and fully active rPN-1 for structural and functional studies.

Keywords: Protease Nexin-1; Thrombin; Neuroblastoma NB2A cell line; Conformational characterization; Recombinant protein

A proteomic approach to characterizing ciglitazone-induced cancer cell differentiation in Hep-G2 cell line by Patrizia Bottoni; Bruno Giardina; Alberto Vitali; Alma Boninsegna; Roberto Scatena (pp. 615-626).

Drug induced cell differentiation represents a promising experimental model for proteomic analysis of cancer cells. In fact, by modulating and monitoring neoplastic cell differentiation it could be possible to identify cytodifferentiation related protein expression changes that can be subsequently utilized in vivo as potential cancer biomarkers. One main advantage of this approach is the significant reduction of biological variability normally observed in clinical biomarker research, with important implications also in prognosis and therapy. At this regard, a new class of differentiating agents is emerging, the so called PPAR-ligands, which however are characterized by a debated mechanism of action that has not been yet studied through a proteomic approach. To this aim, we investigated ciglitazone-induced differentiation of a human hepatocarcinoma HepG2 cell line, by monitoring biochemical and cellular parameters of cytodifferentiation and modifications of cellular protein profiles through 2-DE and MALDI-TOF analysis. Independent of the hypothesized mechanism of action of this intriguing PPARγ agonist, results indicated that ciglitazone is a strong differentiating agent for the HepG2 cell line and that this process is associated with modifications of protein expression related to cell antioxidant systems, the cell cycle apparatus, signal transduction pathways, cellular stress and invasiveness. At last, considering these and other published data, a proteomic profile related to the cancer aggressiveness is beginning to emerge.

Keywords: Biomarker; Cancer; Cell differentiation; Diagnostics; Oncoproteomics; Mitochondria; Stress protein

Structural characterization of the protein cce_0567 from Cyanothece 51142, a metalloprotein associated with nitrogen fixation in the DUF683 family by Garry W. Buchko; Howard Robinson; Anthony Addlagatta (pp. 627-633).

The genomes of many cyanobacteria contain the sequence for a small protein with a common “Domain of Unknown Function” grouped into the DUF683 protein family. While the biological function of DUF683 is still not known, their genomic location within nitrogen fixation clusters suggests that DUF683 proteins may play a role in the process. The diurnal cyanobacterium Cyanothece sp. PCC 51142 contains a gene for a protein that falls into the DUF683 family, cce_0567 (78 aa, 9.0 kDa). In an effort to elucidate the biochemical role DUF683 proteins may play in nitrogen fixation, we have determined the first crystal structure for a protein in this family, cce_0567, to 1.84 Å resolution. Cce_0567 crystallized in space group P2₁ with two protein molecules and one Ni²⁺ cation per asymmetric unit. The protein is composed of two α-helices, residues P11 to G41 (α1) and L49–E74 (α2), with the second α-helix containing a short 3₁₀-helix (Y46–N48). A four-residue linker (L42–D45) between the helices allows them to form an anti-parallel bundle and cross over each other towards their termini. In solution it is likely that two molecules of cce_0567 form a rod-like dimer by the stacking interactions of ∼1/2 of the protein. Histidine-36 is highly conserved in all known DUF683 proteins and the N2 nitrogen of the H36 side chain of each molecule in the dimer is coordinated with Ni²⁺ in the crystal structure. The divalent cation Ni²⁺ was titrated into¹⁵N-labeled cce_0567 and chemical shift perturbations were observed only in the¹H–¹⁵N HSQC spectra for residues at, or near, the site of Ni²⁺ binding observed in the crystal structure. There was no evidence for an increase in the size of cce_0567 upon binding Ni²⁺, even in large molar excess of Ni²⁺, indicating that a metal was not required for dimer formation. Circular dichroism spectroscopy indicated that cce_0567 was extremely robust, with a melting temperature of ∼62 °C that was reversible.

Keywords: Metalloprotein; Nitrogen fixation; Cyanobacteria; Circadian rhythm; Nickel-binding

Altered chain-length and glycosylation modify the pharmacokinetics of human serum albumin by Yasunori Iwao; Mikako Hiraike; Ulrich Kragh-Hansen; Keiichi Kawai; Ayaka Suenaga; Toru Maruyama; Masaki Otagiri ⁎ (pp. 634-641).

Human serum albumin with modified plasma half-life will be useful for clinical purposes. Therefore, the pharmacokinetics of three of each of the following types of genetic variants, and of their corresponding normal albumin, were examined in mice: N-terminally elongated, C-terminally truncated and glycosylated albumins. Isoforms differing from the normal protein by three or more amino acids, especially two of the truncated forms, had shorter half-lives. The effect of glycosylation depended on the position of attachment: in domain II it increased half-life, whereas in domain I and III it had no significant effect. Liver, kidney and spleen uptake clearances were also modified. The pronounced changes in half-life of the two truncated variants and the glycosylated isoform could be explained, at least partly, by large changes in organ uptakes; in the remaining six cases, different effects were registered. Such information should be useful when designing therapeutical albumin products for, e.g., drug delivery systems. In addition to various types of cell endocytosis, leading to intracellular destruction or recycling of the proteins, the metabolism of the alloalbumins could be affected by plasma enzymes. No correlation was found between mutation-induced changes in the pharmacokinetic parameters and changes in α-helical content or changes in heat stability as represented by ΔH _v.

Keywords: Abbreviations; HSA; human serum albumin; rHSA; recombinant HSA; Alb; albumin; Alb A; normal (wild-type) albumin; CD; circular dichroism; Δ; H; _v; van't Hoff enthalpy; RAGE; receptor for advanced glycation end productsHuman serum albumin; Genetic variant; Pharmacokinetics; Half-life; Hepatic uptake; Renal disposition; Spleen uptake

Elucidation of the substrate specificity, kinetic and catalytic mechanism of adenylosuccinate lyase from Plasmodium falciparum by Vinay Bulusu; Bharath Srinivasan; Monnanda Ponnappa Bopanna; Hemalatha Balaram ⁎ (pp. 642-654).

Adenylosuccinate lyase (ASL) catalyzes two distinct but chemically similar reactions in purine biosynthesis. The first, exclusive to the de novo pathway involves the cleavage of 5-aminoimidazole-4-(N-succinylcarboxamide) ribonucleotide (SAICAR) to 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and fumarate and the second common to both de novo and the salvage pathways involves the cleavage of succinyl-adenosine monophosphate (SAMP) to AMP and fumarate. A detailed kinetic and catalytic mechanism of the recombinant His-tagged ASL from Plasmodium falciparum (PfASL) is presented here. Initial velocity kinetics, product inhibition studies and transient kinetics indicate a Uni-Bi rapid equilibrium ordered mechanism. Substrate and solvent isotope effect studies implicate the process of C(γ)–N bond cleavage to be rate limiting. Interestingly, the effect of pH on k_cat and k_cat/ K_m highlight ionization of the base only in the enzyme substrate complex and not in the enzyme alone, thereby implicating the pivotal role of the substrate in the activation of the catalytic base. Site-directed mutagenesis implicates a key role for the conserved serine (S298) in catalysis. Despite the absence of a de novo pathway for purine synthesis and most importantly, the absence of other enzymes that can metabolise AICAR in P. falciparum, PfASL catalyzes the SAICAR cleavage reaction with kinetic parameters similar to those of SAMP reaction and binds AICAR with affinity similar to that of AMP. The presence of this catalytic feature allows the use of AICAR or its analogues as inhibitors of PfASL and hence, as novel putative anti-parasitic agents. In support of this, we do see a dose dependent inhibition of parasite growth in the presence of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAriboside) with half-maximal inhibition at 167±5 μM.

Keywords: Abbreviations; ASL; adenylosuccinate lyase; PfASL; Plasmodium falciparum; ASL; DTT; dithiothreitol; IC; ₅₀; 50% inhibitory concentration; SAICAR; 5-aminoimidazole-4-(N-succinylcarboxamide) ribonucleotide; AICAR; 5-aminoimidazole-4-carboxamide ribonucleotide; AICAriboside; 5-aminoimidazole-4-carboxamide ribonucleoside; SAMP; succinyl-adenosine monophosphate; KIE; kinetic isotope effect; ITC; isothermal titration calorimetry Plasmodium falciparum; Adenylosuccinate lyase; Catalysis; Rapid equilibrium; AICAR

Stability improvement of the fatty acid binding protein Sm14 from S. mansoni by Cys replacement: Structural and functional characterization of a vaccine candidate by Celso R.R. Ramos; Alberto Spisni ⁎; Sérgio Oyama Jr.; Mauricio L. Sforça; Henrique R. Ramos; Mônica M. Vilar; Adriana C. Alves; Rita C.R. Figueredo; Míriam Tendler; Nilson I.T. Zanchin; Thelma A. Pertinhez; Paulo Lee Ho ⁎ (pp. 655-662).

The Schistosoma mansoni fatty acid binding protein (FABP), Sm14, is a vaccine candidate against, S. mansoni and F. hepatica. Previously, we demonstrated the importance of a correct fold to achieve protection in immunized animals after cercariae challenge [[10]. C.R.R. Ramos, R.C.R. Figueredo, T.A. Pertinhez, M.M. Vilar, A.L.T.O. Nascimento, M. Tendler, I. Raw, A. Spisni, P.L. Ho, Gene structure and M20T polymorphism of the Schistosoma mansoni Sm14 fatty acid-binding protein: structural, functional and immunoprotection analysis. J. Biol. Chem. 278 (2003) 12745–12751.]. Here we show that the reduction of vaccine efficacy over time is due to protein dimerization and subsequent aggregation. We produced the mutants Sm14-M20(C62S) and Sm14-M20(C62V) that, as expected, did not dimerize in SDS-PAGE. Molecular dynamics calculations and unfolding experiments highlighted a higher structural stability of these mutants with respect to the wild-type. In addition, we found that the mutated proteins, after thermal denaturation, refolded to their active native molecular architecture as proved by the recovery of the fatty acid binding ability. Sm14-M20(C62V) turned out to be the more stable form over time, providing the basis to determine the first 3D solution structure of a Sm14 protein in its apo-form. Overall, Sm14-M20(C62V) possesses an improved structural stability over time, an essential feature to preserve its immunization capability and, in experimentally immunized animals, it exhibits a protection effect against S. mansoni cercariae infections comparable to the one obtained with the wild-type protein. These facts indicate this protein as a good lead molecule for large-scale production and for developing an effective Sm14 based anti-helminthes vaccine.

Keywords: Abbreviations; CD; circular dichroism; DAUDA; 11-(dansylamino) undecanoic acid; FABP; fatty acid binding protein; F. hepatica; Fasciola hepatica; MD; molecular dynamics; MPL; monophosphoryl lipid A; PBS; phosphate-buffered saline; Rg; radius of gyration; RMSD; root mean square deviation; S. mansoni; Schistosoma mansoni; SDS-PAGE; sodium dodecyl sulfate poliacrylamide gel electrophoresis; TDM; trehalose dicorynomycolate; WHO/TDR; World Health Organization/Special Program for Research and Training in Tropical DiseasesNuclear Magnetic Resonance; Sm14; Vaccine; Schistosoma mansoni; Schistosomiasis; Fatty-acid binding protein

Rapid elevation of Inos and decreases in abundance of other proteins at pupal diapause termination in the flesh fly Sarcophaga crassipalpis by Aiqing Li; M. Robert Michaud; David L. Denlinger ⁎ (pp. 663-668).

We analyzed changes in brain proteins 24 h after pupal diapause termination in Sarcophaga crassipalpis by a combination of 2-dimensional gel electrophoresis and mass spectrometry. The proteome analysis revealed significant changes in 20 proteins, 11 of which represented ≥2.5-fold changes. Three proteins were present only in the brains of diapausing pupae. Among the most abundant proteins that showed a change, 1 was more abundant, 7 were less abundant, and 2 were absent following diapause termination. The protein that increased in abundance following diapause termination showed highest identity to myo-inositol-1-phosphate synthase (Inos). Proteins that decreased at diapause termination included those showing highest identities to fatty acid binding protein, CG2331-PA, twinstar, catalase, and a histone. Proteins absent at diapause termination included ribosomal protein L17A and one unnamed protein. An increase of Inos protein level was confirmed using Western blot analysis. Attempts to terminate diapause by injection of several Inos-related metabolites failed, thus suggesting that the elevation of Inos at diapause termination is downstream of the physiological regulation that initiates development.

Keywords: Proteomics; Diapause; Diapause termination; Brain protein; Sarcophaga crassipalpis

Cold destabilization and temperature jump of the murine prion protein mPrP(23–231) by Tomoharu Matsumoto; Tatsuo Nakagawa; Kazuo Kuwata ⁎ (pp. 669-673).

We analyzed the thermal stability of the recombinant murine prion protein mPrP(23–231) with a single tryptophan mutation (F174W) and its perturbation by cold temperature. Compared to the N-terminally truncated ones, full-length construct is significantly unstable and forms intermediate state of urea denaturation, and also undergoes the cold destabilization under the ambient pressure. In order to detect the very early phase of the folding, we also applied a laser-induced temperature jump kinetic measurement and observed a kinetic phase of several microseconds, suggesting the barrierless folding process. The conformational instability and low barriers between different conformers may explain the unusual flexibility leading to the pathogenic conversion and the strain diversity.

Keywords: Abbreviations; PrP; prion protein; ID; ₅₀; median infectious dose; cT-jump; continuous-wave probed laser-induced temperature jump; CD; circular dichroism; NATA; N; -acetyltryptophanamidePrion protein; Folding intermediate; Cold denaturation; Structural stability; Laser-induced temperature jump

A novel vanadium reductase, Vanabin2, forms a possible cascade involved in electron transfer by Norifumi Kawakami; Tatsuya Ueki; Yusuke Amata; Kan Kanamori; Koichi Matsuo; Kunihiko Gekko; Hitoshi Michibata ⁎ (pp. 674-679).

The unusual ascidian ability to accumulate high levels of vanadium ions at concentrations of up to 350 mM, a 10⁷-fold increase over that found in seawater, has been attracting interdisciplinary attention for a century. Accumulated V^V is finally reduced to V^III via V^IV in ascidian vanadocytes. Reducing agents must therefore participate in the reduction. Previously, we identified a vanadium-binding protein, Vanabin2, in which all 18 cysteines form nine disulfide bonds. Here, we report that Vanabin2 is a novel vanadium reductase because partial cleavage of its disulfide bonds results in the reduction of V^V to V^IV. We propose that Vanabin2 forms a possible electron transfer cascade from the electron donor, NADPH, via glutathione reductase, glutathione, and Vanabin2 to the acceptor, and vanadium ions conjugated through thiol–disulfide exchange reactions.

Keywords: Ascidian; Vanadium; Vanabin2; Redox; Thiol–disulfide exchange reaction

Exploring the structural and functional stabilities of different paraoxonase-1 formulations through electrophoretic mobilities and enzyme activity parameters under hydrostatic pressure by Cécile Cléry-Barraud; Frédérique Renault; Julien Leva; Nacéra El Bakdouri; Patrick Masson; Daniel Rochu ⁎ (pp. 680-688).

Human paraoxonase-1 (HuPON1) is the ideal candidate to engineer as catalytic bioscavenger for pre-treatment and therapy of exposure to toxic organophosphorus compounds. HuPON1 is a naturally-occurring hydrophobic plasma protein associated with a partner, the human phosphate binding protein (HPBP) on high density lipoproteins. The relationships between the composition and the size of multimeric states of HuPON1 are not well understood. Moreover, the effect of HPBP's presence on enzyme catalysis and stability is not clear. The effect of hydrostatic pressure on structural stability and activity of different PON1 preparations (free natural HuPON1 or in the presence of 50% w/w HPBP, hybrid recombinant PON1) was investigated. Results showed that PON1 exists under several multimeric forms, and that the binding of HPBP amends the size of the hetero-oligomeric states and exerts a stabilizing effect on the activities of PON1. Furthermore, high pressure kinetic experiments highlighted the fact that PON1 displays two distinct catalytic behaviors: the first one for arylesterase and lactonase activities and the second one for its organophosphate-hydrolase activity.

Keywords: Abbreviations; CE; capillary electrophoresis; Coum; 2-coumaranone; FP; Ferguson plot; HDL; high-density lipoprotein; HHP; high hydrostatic pressure; HPBP; human phosphate binding protein; HuPON1; human paraoxonase-1; HuPON1–HPBP (9:1); HuPON1 preparation in the presence of 10% HPBP; HuPON1–HPBP (1:1); HuPON1 preparation in the presence of 50% HPBP; OP; organophosphorus compound; P; _atm; atmospheric pressure; PhA; phenyl acetate; Pox; paraoxon; rePON1; recombinant paraoxonase-1Paraoxonase; Human phosphate binding protein; High hydrostatic pressure; Oligomerization; Stability; Functional stability

New oxidase from Bjerkandera arthroconidial anamorph that oxidizes both phenolic and nonphenolic benzyl alcohols by Elvira Romero; Patricia Ferreira; Ángel T. Martínez; María Jesús Martínez (pp. 689-697).

A new flavooxidase is described from a Bjerkandera arthroconidial anamorph. Its physicochemical characteristics, a monomeric enzyme containing non-covalently bound flavin adenine dinucleotide (FAD), and several catalytic properties, such as oxidation of aromatic and polyunsaturated aliphatic primary alcohols, are similar to those of Pleurotus eryngii aryl-alcohol oxidase (AAO). However, it also efficiently oxidizes phenolic benzyl and cinnamyl alcohols that are typical substrates of vanillyl-alcohol oxidase (VAO), a flavooxidase from a different family, characterized by its multimeric nature and presence of covalently-bound FAD. The enzyme also differs from P. eryngii AAO by having extremely high efficiency oxidizing chlorinated benzyl alcohols (1000–1500 s−¹ mM−¹), a feature related to the different alcohol metabolites secreted by the Pleurotus and Bjerkandera species including chloroaromatics, and higher activity on aromatic aldehydes. What is even more intriguing is the fact that, the new oxidase is optimally active at pH 6.0 on both p-anisyl and vanillyl alcohols, suggesting a mechanism for phenolic benzyl alcohol oxidation that is different from that described in VAO, which proceeds via the substrate phenolate anion formed at basic pH. Based on the above properties, and its ADP-binding motif, partially detected after N-terminus sequencing, the new enzyme is classified as a member of the GMC (glucose–methanol–choline oxidase) oxidoreductase family oxidizing both AAO and VAO substrates.

Keywords: Abbreviations; AAD; aryl-alcohol dehydrogenase; AAO; aryl-alcohol oxidase; ABTS; 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); Endo H; endoglycosidase H; FAD; flavin adenine dinucleotide; GMC; glucose–methanol–choline oxidase; HRP; horseradish peroxidase; k; _cat; maximal turnover number; K; _i; inhibition constant; K; _m; Michaelis constant; MALDI; matrix-assisted laser desorption/ionization; p; I; isoelectric point; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TOF; time of flight; U; enzyme unit; VAO; vanillyl-alcohol oxidaseFlavoenzyme; Benzyl alcohol; Hydroxybenzyl alcohol; Aromatic aldehyde; Aryl-alcohol oxidase; Vanillyl-alcohol oxidase

Resistance of bromelain to SDS binding by Reema Bhattacharya; Debasish Bhattacharyya ⁎ (pp. 698-708).

Interaction of the plant cysteine protease bromelain with SDS has been studied using CD spectroscopy, intrinsic fluorescence emission, extrinsic fluorescence probe pyrene, isothermal calorimetric (ITC) investigations and inhibition of hydrolyzing activity. Results exhibit number of synchronous transitions when plotted against the total SDS concentration. SDS at submicellar level caused conformation change of bromelain leading to a stable entity. ITC and pyrene experiments suggest that the structural modifications below 5 mM, the cmc_app of SDS solutions containing bromelain, are the result of alterations of solvent hydrophobicity or non-specific weak binding and/or adsorption of SDS monomers. Melting temperature ( T_m) and the free energy change for thermal unfolding (Δ G_unf) of the SDS induced conformers was decreased by 5 °C and 0.5 kcal/mol respectively, compared to native bromelain. Below 5 mM, SDS caused large decrease in V_max without affecting K_m for the substrate Z-Arg-Arg-NHMec. Analysis of kinetic data imply that SDS acts as a partial non-competitive inhibitor since even at 100 mM, the residual activity of bromelain was retained by 3%. Inhibition studies show an IC₅₀ of 0.55 mM and a high K_i of 0.145 mM. These demonstrate that bromelain is resistant to SDS binding and denaturation, a property known for β-sheet rich kinetically stable proteins.

Keywords: Abbreviations; MRE; mean residue ellipticity; cmc; critical micelle concentrationBromelain; Isothermal calorimetry; Resistance to SDS; Partial inhibition; Protein stability

Molecular interaction between apo or holo α-lactalbumin and lysozyme: Formation of heterodimers as assessed by fluorescence measurements by Michaël Nigen; Véronique Le Tilly; Thomas Croguennec; Delphine Drouin-Kucma; Saïd Bouhallab (pp. 709-715).

In a previous work, we reported that contrary to native calcium-loaded α-lactalbumin (holo α-LA), calcium-depleted form (apo α-LA) has the ability to self-assemble with lysozyme (LYS) to form different supramolecular structures in temperature-dependent manner. In this study, we examine what happens at molecular scale using fluorescence techniques. Fluorescence anisotropy coupled with fluorescence lifetime measurements provides a means to measure intermolecular interactions. We showed that LYS interacts with both apo α-LA and holo α-LA to form oligomers, assumed to be heterodimers, at 10 °C and 45 °C. The dissociation constants for dimerization were found to be in the μM range and increased significantly with increasing ionic strength from 39 to 124 mM. Although the binding constants of holo α-LA–LYS and apo α-LA–LYS complexes were of the same order of magnitude, the shape or conformation of formed heterodimers differed as assessed by fluorescence parameters in particular correlation time calculations. Such conformation differences could explain why holo α-LA–LYS complexes are trapped as heterodimers while the apo α-LA–LYS complexes have the ability to further self-assemble into various supramolecular structures.

Keywords: α-Lactalbumin; Lysozyme; Interaction; Heterodimer; Fluorescence

Structural and mechanistic analyses of yeast mitochondrial thioredoxin Trx3 reveal putative function of its additional cysteine residues by Rui Bao; Yaru Zhang; Cong-Zhao Zhou; Yuxing Chen ⁎ (pp. 716-721).

The yeast Saccharomyces cerevisiae Trx3 is a key member of the thioredoxin system to control the cellular redox homeostasis in mitochondria. We solved the crystal structures of yeast Trx3 in oxidized and reduced forms at 1.80 and 2.10 Å, respectively. Besides the active site, the additional cysteine residue Cys69 also undergoes a significant redox-correlated conformational change. Comparative structural analyses in combination with activity assays revealed that residue Cys69 could be S-nitrosylated in vitro. S-nitrosylation of Cys69 will decrease the activity of Trx3 by 20%, which is comparable to the effect of the Cys69Ser mutation. Taken together, these findings provided us some new insights into the putative function of the additional cysteine residues of Trx3.

Keywords: Thioredoxin; Saccharomyces cerevisiae; Crystal structure; Mitochondrion; S-nitrosylation

Corrigendum to “No effect of covalently linked poly(ethylene glycol) chains on protein internal dynamics” [Biochem. Biophys. Acta 1794 (2009) 569–576] by Margherita Gonnelli; Giovanni B. Strambini ⁎ (pp. 722-722).

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BBA - Proteins and Proteomics (v.1794, #4)