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BBA - Proteins and Proteomics (v.1774, #1)

A word of farewell and a word of welcome (pp. 1-2).

BBA in the year 2007 by Dennis E. Vance Editor-in-Chief, BBA (pp. 3-4).

Intramural delivery of Sirolimus prevents vascular remodeling following balloon injury by Michael Buerke; Markus Guckenbiehl; Hansjörg Schwertz; Ute Buerke; Michael Hilker; Herbert Platsch; Joachim Richert; Sabine Bomm; Guy A. Zimmerman; Stephan Lindemann; Ursula Mueller-Werdan; Karl Werdan; Harald Darius; Andrew S. Weyrich (pp. 5-15).

Objective. Several studies have demonstrated that Sirolimus-eluting stents reduce restenosis in patients with coronary artery disease. Here, we tested whether direct delivery of Sirolimus into the vessel wall during balloon angioplasty can modify vascular remodeling over several weeks. Methods and Results. During angioplasty of the rabbit iliac artery we administered an intramural infusion of Sirolimus or its vehicle directly through a balloon catheter into the vessel wall. After 3 weeks neointimal formation was decreased (0.71±0.1 vs. 1.4±0.12 intima/media ratio), and this process was attributed to the inhibitory properties of Sirolimus on ECM deposition and smooth muscle cell proliferation. Sirolimus also significantly reduced the deposition of elastin, collagen III and fibronectin within the vascular wall. In parallel, proteomic profiles of arterial wall segments were obtained and 485 protein spots were consistently matched between non-dilated and dilated vessels. Differential expression of 12 proteins were observed between the groups and direct sequencing of digested peptides was performed. Local delivery of sirolimus during angioplasty attenuated the expression of structural proteins that included lamin A, vimentin, α-1-antitrypsin, and α-actin. Conclusions. Local administration of Sirolimus during angioplasty prevents smooth muscle cell proliferation associated with vascular remodeling as well as the expression of extracellular matrix and structural proteins. Therefore, local injection of Sirolimus during balloon inflation may be an alternative therapeutic approach for preventing restenosis in small stenotic vessels (i.e., <2.5 mm).

Keywords: Abbreviations; ECM; extracellular matrix; PDGF; platelet derived growth factor; PTA; percutaneous transluminal angioplasty; VSMC; vascular smooth muscle cells; FKBP12; FK506-binding protein 12; I/M-ratio; intima/media ratio; 2D PAGE; Two-dimensional Gel Electrophoresis; FCS; fetal calf serumAngioplasty; Neointima formation; Sirolimus; 2D-proteome analysis; Vascular injury

Hydrolysis of oxo- and thio-esters by human butyrylcholinesterase by Patrick Masson; Marie-Thérèse Froment; Emilie Gillon; Florian Nachon; Oksana Lockridge; Lawrence M. Schopfer (pp. 16-34).

Catalytic parameters of human butyrylcholinesterase (BuChE) for hydrolysis of homologous pairs of oxo-esters and thio-esters were compared. Substrates were positively charged (benzoylcholine versus benzoylthiocholine) and neutral (phenylacetate versus phenylthioacetate). In addition to wild-type BuChE, enzymes containing mutations were used. Single mutants at positions: G117, a key residue in the oxyanion hole, and D70, the main component of the peripheral anionic site were tested. Double mutants containing G117H and mutations on residues of the oxyanion hole (G115, A199), or the π-cation binding site (W82), or residue E197 that is involved in stabilization of tetrahedral intermediates were also studied. A mathematical analysis was used to compare data for BuChE-catalyzed hydrolysis of various pairs of oxo-esters and thio-esters and to determine the rate-limiting step of catalysis for each substrate. The interest and limitation of this method is discussed. Molecular docking was used to analyze how the mutations could have altered the binding of the oxo-ester or the thio-ester. Results indicate that substitution of the ethereal oxygen for sulfur in substrates may alter the adjustment of substrate in the active site and stabilization of the transition-state for acylation. This affects the k₂/ k₃ ratio and, in turn, controls the rate-limiting step of the hydrolytic reaction. Stabilization of the transition state is modulated both by the alcohol and acyl moieties of substrate. Interaction of these groups with the ethereal hetero-atom can have a neutral, an additive or an antagonistic effect on transition state stabilization, depending on their molecular structure, size and enantiomeric configuration.

Keywords: Abbreviations; AChE; Acetylcholinesterase; ACh; acetylcholine; ASCh; acetylthiocholine; BuChE; butyrylcholinesterase; BuCh; butyrylcholine; BuSCh; butyrylthiocholine; BzCh; benzoylcholine; BzSCh; benzoylthiocholine; CPO; chlorpyrifos-oxon; DFP; diisopropylfluorophosphate; OP; organophosphate; PAS; peripheral anionic site; PhA; phenylacetate; PhSA; phenylthioacetate; PrCh; propionylcholine; PrSCh; propionylthiocholine; SdCh; succinyldicholine; SdSCh; succinyldithiocholineButyrylcholinesterase; Ester; Thio-ester; Oxyanion hole; Mutation; Rate-limiting step

Solution structure of the second SH3 domain of human CMS and a newly identified binding site at the C-terminus of c-Cbl by Bo Yao; Jiahai Zhang; Haiming Dai; Jianping Sun; Yuanyuan Jiao; Yajun Tang; Jihui Wu; Yunyu Shi (pp. 35-43).

CMS, cas ligand with multiple Src homology 3 (SH3) domains, belongs to a family of ubiquitously expressed adaptor proteins. Among the CMS binding proteins, c-Cbl has been mostly extensively studied. It was reported that the motif PKPFPR (residues 824–829) of c-Cbl can bind to the N-terminus SH3 domains of CMS. Here we report the solution structure of the second SH3 domain of CMS (CMS_SH3_B), furthermore, we have identified that a peptide from residues 701 to 714 of c-Cbl (Cbl-p), i.e. MTPSSRPLRPLDTS, can specially bind to CMS_SH3_B using NMR chemical shift perturbation, suggesting that the peptide is a new potential CMS binding site. Among the peptide, TPSSRPLR is the core binding motif and Arg709 plays a key role in the interaction. Cbl-p binding interface on CMS_SH3_B along a hydrophobic channel is composed of RT loop, n-Src loop and β4 strand and divided into three pockets. This work indicates the solution structure of CMS_SH3_B bears the canonical β–β–β–β–α–β fold and a new binding site in c-Cbl involved in its interaction with CMS, which probably contributes to the clustering of CMS. All the information provided here should be beneficial for the future functional study of CMS.

Keywords: Abbreviations; 2D and 3D; two- and three-dimensional; NMR; nuclear magnetic resonance; HSQC; heteronuclear single quantum correlation; NOE; nuclear Overhauser effect; NOESY; nuclear Overhauser effect spectroscopy; TOCSY; total correlated spectroscopy; RMSD; root-mean-square deviation; CMS; cas ligand with multiple Src homology 3 (SH3) domainsCMS; SH3 domain; C-Cbl; Binding site; NMR

Proteome analysis of Halobacterium salinarum and characterization of proteins related to the degradation of isopropyl alcohol by Dong-Jin Ha; Won-A Joo; Gi-Yeon Han; Chan-Wha Kim (pp. 44-50).

We reported in a previous study that proteomic approach, coupled with genomic techniques, could be used to screen and develop multiple candidates for halophilic enzymes from Halobacterium salinarum. In order to evaluate the biodegradation of isopropyl alcohol (IPA) by H. salinarum, the amounts of residual IPA and acetone generated in the growth media were determined using a gas chromatography-flame ionization detector (GC-FID). The protein expression profiles of cells which had been cultured with IPA were obtained with the two-dimensional gel electrophoresis. Proteins evidencing different expression levels in the presence of 0.5% IPA were identified by electrospray ionization-quadruple-time of flight (ESI-Q-TOF) mass spectrometry. We found 12 proteins which were down-regulated, and another 12 proteins which were up-regulated, in the presence of 0.5% IPA and we further identified 17 proteins among them using ESI-TOF MS/MS. Among these identified proteins, we selected glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for further characterization as a halophilic enzyme. We have demonstrated for the first time that H. salinarum possesses the ability to degrade IPA and GAPDH was both stable and active at high salt concentrations, with maximum activity occurring at 1 M NaCl, although the optimal salt concentration with regard to the growth of H. salinarum is 4.3 M.

Keywords: Halobacterium salinarum; GC-FID; 2-DE; ESI-Q-TOF; GAPDH

Interaction of rhein with human serum albumin investigation by optical spectroscopic technique and modeling studies by Ying Li; Xiaojun Yao; Jing Jin; Xingguo Chen; Zhide Hu (pp. 51-58).

The binding of rhein with human serum albumin (HSA) has been studied in detail by spectroscopic method including circular dichroism (CD), Fourier transformation infrared spectra (FT-IR), fluorescence spectra. The binding parameters for the reaction have been calculated according to Scatchard equation at different temperatures. The plots indicated that the binding of HSA to rhein at 303, 310 and 318 K is characterized by one binding site with the affinity constant K at (4.93±0.16)×10⁵, (4.02±0.16)×10⁵ and (2.69±0.16)×10⁵ M^-1, respectively. The secondary structure compositions of free HSA and its rhein complexes were estimated by the FT-IR spectra. FT-IR and curve-fitted results of amide I band are in good agreement with the analyses of CD spectra. Molecular Modeling method was used to calculate the interaction modes between the drug and HSA.

Keywords: Human serum albumin (HSA); Rhein; Circular dichroism (CD); Fourier transformation infrared spectra (FT-IR); Fluorescence quenching; Fluorescence anisotropy

Structural basis for the protective role of sulfite against transthyretin amyloid formation by Luís Gales; Maria J. Saraiva; Ana M. Damas (pp. 59-64).

Transthyretin (TTR) is a plasma protein, which under conditions not yet completely understood, aggregates forming amyloid deposits that occur extracellularly. It is a protein composed of four identical subunits. Each monomer has a single cysteine residue (Cys10), which in the plasma is reduced (Cys-SH), oxidized (Cys-SO₃⁻), sulfonated (Cys-S-SO₃⁻) or bound to various sulfhydryls. There is evidence that these chemical modifications of the SH group alter the stability and the amyloidogenic potential of the protein. The sulfonated form was found to enhance the stability of the native conformation of TTR, avoiding misassembly of the protein leading to amyloid. Consequently, the potential treatment of TTR-type amyloidosis by sulfite has been suggested.The structure of TTR pre-incubated with sulfite at physiological pH, was determined by X-ray crystallography to provide structural insight for the stabilizing effect of sulfite. Each subunit has a β-sandwich conformation, with two four stranded β-pleated sheets (DAGH and CBEF) and a small α-helix between strands. The sulfonated cysteines have two sulfite oxygens involved in intramonomer hydrogen bonds that bridge Cys10, the amino acid immediately before β-strand A, to the amino acids immediately after the edge β-strand D. Implications of the newly observed interactions in the inhibition of fibril formation are discussed in light of the recent structural models of TTR amyloid fibrils.

Keywords: Amyloid; Sulfite; Transthyretin; Familial amyloidotic polyneuropathy; X-ray crystallography

N-Ethylmaleimide-resistant acyl-coenzyme A oxidase from Arthrobacter ureafaciens NBRC 12140: Molecular cloning, gene expression and characterization of the recombinant enzyme by Mikio Bakke; Chiaki Setoyama; Retsu Miura; Naoki Kajiyama (pp. 65-71).

N-Ethylmaleimide (NEM)-resistant acyl-coenzyme A oxidase (ACO) has been desired for the determination of free fatty acids (FFAs). In order to meet this demand, we prepared recombinant ACO from Arthrobacter ureafaciens NBRC 12140. The coding region of the gene was 2109, encoding a protein of 703 amino acids with a predicted molecular mass of 76.5 kDa. The heterologous expression level in Escherichia coli was 520-fold higher than that in the native strain. The purified enzyme retained more than 60% activity after incubation in the presence of 10 mM NEM at 37 °C for 4 h, while other commercially available ACOs showed only less than 10% activities after the same NEM treatment. We presume that this is due to the presence of only three cysteines in ACO from A. ureafaciens. Site-directed mutagenesis studies and close scrutiny of the three-dimensional structures of other related ACOs suggested that these cysteines were buried in the protein and unreactive to NEM. The recombinant enzyme was used for the colorimetric determination of free fatty acid, which gave a linear calibration.

Keywords: Acyl-CoA oxidase; Arthrobacter; sp.; N; -ethylmaleimide-resistance; Cloning; Heterologous expression; Site-directed mutagenesis

Biophysical investigation of recombinant K5 lyase: Structural implications of substrate binding and processing by Angelika Rek; James Thompson; Ian S. Roberts; Andreas J. Kungl (pp. 72-77).

K5 lyase of coliphage K5A degrades the K5 polysaccharide of encapsulated E. coli strains expressing the K5 antigen thereby contributing to virus binding and infection. We have investigated the affinities of the recombinant enzyme for different GAG ligands by isothermal fluorescence titrations and correlated them with substrate processing and protein structural changes. Chondroitin sulfate (CS) and heparan sulfate (HS) bound to K5 lyase with a K_d of 0.5 μM whereas heparin exhibited a K_d=1.1 μM. The natural substrate K5 polysaccharide displayed a similar apparent affinity as CS and HS but was the only ligand of the enzyme which induced a large structural rearrangement of the protein as detected by far-UV CD spectroscopy. Since significant enzymatic degradation was only found for the K5 polysaccharide peaking at 44 °C, but binding was also detected for heparin, we propose that the K5 lyase is able to discriminate between specific (acetylated/non-sulfated) and unspecific (acetylated/sulfated) ligands by its heparin binding motif in the C-terminus. This is proposed to be the origin for the enzyme's residual HS degrading activity.

Keywords: Abbreviations; CD; circular dichroism; dp; degree of polymerisation; GAGs; glycosaminoglycans; GlcA; glucuronic acid; GlcNAc; N-acetyl-glucosamin; HS; heparan sulfate; HA; hyaluronic acid; CS; chondroitin sulfate; K; _d; dissociation constant; PBS; phosphate buffered salineCarbohydrate; Circular dichroism; Fluorescence; Glycosaminoglycan; K5 lyase

A truncated peptide model of the mutant P61A FIS forms a stable dimer by Daniel F. Moriarty; Christine Fiorillo; Charmi Miller; Wilfredo Colón (pp. 78-85).

Factor for inversion stimulation (FIS) is a 98-residue homodimeric DNA-binding protein involved in several different cellular processes including DNA inversion and the regulation of multiple genes. FIS contains a flexible and functionally important N-terminus followed by four helices (A–D), the last two of which consist of the DNA-binding region. Helix B, which comprises the main dimerization interface has a 20° kink at its center that was originally thought to be caused by the presence of a proline at position 61. However, it was later shown that the kink remained largely intact and that FIS retained its native-like function when the proline was mutated to an alanine. We previously showed that the P61A mutation increased the stability of FIS, but decreased its equilibrium denaturation cooperativity apparently due to preferential stabilization of the B-helix. Here we studied a peptide of P61A FIS, corresponding to residues 26–71 (26–71(A3) FIS), which encompasses the dimer interface (helices A and B). Circular dichroism (CD) and size-exclusion chromatography/multi-angle light scattering showed that the peptide was α-helical and dimeric, respectively, as expected based on the 3D structure of FIS. Urea-induced equilibrium denaturation experiments monitored by far-UV CD revealed a concentration-dependent transition, and data analysis based on a N₂⇆2U model yielded a Δ G of approximately −10 kcal/mol. Our results suggest that 26–71(A3) FIS can form a stable dimeric structure despite lacking the N- and C-terminus of native FIS.

Keywords: Abbreviations; CD; circular dichroism; EDTA; ethylenediamine-tetraacetic acid; FIS; Factor for inversion stimulation; HPLC; high pressure liquid chromatography; HTH; helix–turn–helix; MALDI-TOF; matrix-assisted laser desorption ionization time of flight; MALS; multi-angle light scattering; Tris–HCl; tris hydroxymethyl aminomethane hydrochloride; WT; wild typeFactor for inversion stimulation; FIS; Circular dichroism; Multi-angle light scattering; HPLC; Equilibrium denaturation

Inactivation of alcohol dehydrogenase (ADH) by ferryl derivatives of human hemoglobin by Aleksandra Kowalczyk; Mieczysław Puchała; Katarzyna Wesołowska; Eligiusz Serafin (pp. 86-92).

In this paper, inactivation of alcohol dehydrogenase (ADH) by products of reactions of H₂O₂ with metHb has been studied. Inactivation of the enzyme was studied in two systems corresponding to two kinetic stages of the reaction. In the first system H₂O₂ was added to the mixture of metHb and ADH [the (metHb+ADH)+H₂O₂] system (ADH was present in the system since the moment of addition of H₂O₂ i. e. since the very beginning of the reaction of metHb with H₂O₂). In the second system ADH was added to the system 5 min after the initiation of the reaction of H₂O₂ with metHb [the (metHb+H₂O₂)_5 min+ADH] system. In the first case all the products of reaction of H₂O₂ with metHb (non-peroxyl and peroxyl radicals and non-radical products, viz. hydroperoxides and *HbFe(IV)=O) could react with the enzyme causing its inactivation. In the second system, enzyme reacted almost exclusively with non-radical products (though a small contribution of reactions with peroxyl radicals cannot be excluded). ADH inactivation was observed in both system. Hydrogen peroxide alone did not inactivate ADH at the concentrations employed evidencing that enzyme inactivation was due exclusively to products of reaction of H₂O₂ with metHb. The rate and extent of ADH inactivation were much higher in the first than in the second system. The dependence of ADH activity on the time of incubation with ferryl derivatives of Hb can be described by a sum of three exponentials in the first system and two exponentials in the second system. Reactions of appropriate forms of the ferryl derivatives of hemoglobin have been tentatively ascribed to these exponentials. The extent of the enzyme inactivation in the second system was dependent on the proton concentration, being at the highest at pH 7.4 and negligible at pH 6.0. The reaction of H₂O₂ with metHb resulted in the formation of cross-links of Hb subunits (dimers and trimers). The amount of the dimers formed was much lower in the first system i. e. when the radical forms dominated the reaction of inactivation.

Keywords: Abbreviations; ADH; alcohol dehydrogenase; metHb; methemoglobin; *metHb; metHb obtained after spontaneous autoreduction of ferrylHb; ferrylHb; ferrylhemoglobin; HbFe(IV); ^⁺; =; O; ^{cation radical of the ferryl derivative of hemoglobin}; HbFe(IV); =; O; radical forms of human ferrylhemoglobin; *HbFe(IV); =; O; non-radical forms of human ferrylhemoglobin; HAS; human serum albuminFerrylhemoglobin; Oxidative defense; Protein radical; Alcohol dehydrogenase; Hydrogen peroxide; Radical transfer

Proteomic analysis of isolated membrane fractions from superinvasive cancer cells by Paul Dowling; Paula Meleady; Andrew Dowd; Michael Henry; Sharon Glynn; Martin Clynes (pp. 93-101).

The superinvasive phenotype exhibited by paclitaxel-selected variants of an in vitro invasive clonal population of the human cancer cell line, MDA-MB-435S were examined using DIGE (Fluorescence 2-D Difference Gel Electrophoresis) and mass spectrometry. Isolation of membrane proteins from the MDA-MB-435S-F/Taxol-10p4p and parental populations was performed by temperature-dependent phase partitioning using the detergent Triton X-114. Subsequent DIGE-generated data analysed using Decyder software showed many differentially-expressed proteins in the membrane fraction. 16 proteins showing statistically significant upregulation in the superinvasive cells were identified by MALDI-ToF. Proteins upregulated in the superinvasive population include Galectin-3, Cofilin, ATP synthase beta subunit, voltage-dependent anion channel 1, voltage dependent anion channel 2, ER-60 protein, MHC class II antigen DR52, Beta actin, TOMM40 protein, Enolase 1, Prohibitin, Guanine nucleotide-binding protein, Annexin II, Heat shock 70 kDa protein, Stomatin-like protein 2 and Chaperonin. Many of these proteins are associated with inhibition of apoptosis, the progression of cancer, tumourigenicity, metastasis, actin remodelling at the leading edge of cells, polarized cell growth, endocytosis, phagocytosis, cellular activation, cytokinesis, and pathogen intracellular motility. These results suggest a correlation between the increased abundance of these proteins with the superinvasive phenotype of the paclitaxel-selected MDA-MB-435S-F/Taxol-10p4p population.

Keywords: Cofilin; DIGE; Galectin-3; Membrane protein; Superinvasive

The effect of dextran on subunit exchange of the molecular chaperone αA-crystallin by Arezou Ghahghaei; Agata Rekas; William E. Price; John A. Carver (pp. 102-111).

α-Crystallin, a member of small heat shock protein (sHsp) family, is comprised of αA and αB subunits and acts as a molecular chaperone by interacting with unfolding proteins to prevent their aggregation. The αA-crystallin homopolymer consists of 30–40 subunits that are undergoing dynamic exchange. In vivo, α-crystallin elicits its chaperone action in a crowded cellular environment (e.g. in the lens). In vitro, inert molecular crowding agents (e.g. dextran) are often used to mimic crowded conditions. In this study, it was found that α-crystallin and αA-crystallin are poorer chaperones in the presence of dextran. Using fluorescence resonance energy transfer, it is shown that the αA-crystallin subunit exchange rate strongly increases with temperature. Binding of reduced ovotransferrin to αA-crystallin markedly decreases the rate of subunit exchange, as does the presence of dextran. In addition, in the presence of dextran the effect of reduced ovotransferrin on decreasing the rate of subunit exchange of αA-crystallin is greater than in the absence of dextran. Under the conditions of molecular crowding, the αA-crystallin subunit exchange rate is not temperature-dependent. In the absence of dextran, the exchange rate of αA-crystallin subunits correlates with its chaperone efficiency, i.e. the chaperone ability of αA-crystallin increases with temperature. However in the presence of dextran, the temperature dependence of the chaperone ability of αA-crystallin is eliminated.

Keywords: Abbreviations; sHsp; (small heat shock protein); FRET; (Fluorescence resonance energy transfer); AIAS; (4-acetamido-4′-((iodoacetyl) amino) stilbene-2,2′-disulfonic acid); LYI; (Lucifer yellow iodoacetamide); DTT; (dithiothreitol)αA-crystallin; Molecular chaperone; Crowding agent; Subunit exchange; FRET

Novel family of cholesterol esterases produced by actinomycetes bacteria by Hongyu Xiang; Shunsuke Masuo; Takayuki Hoshino; Naoki Takaya (pp. 112-120).

Although cholesterol esterase (CHE; EC 3.1.1.13) is widespread in nature, CHEs from Streptomyces lavendulae and Streptomyces sp. X9 are the only known CHEs produced by actinomycetes. We purified CHEs from S. avermitilis JCM5070, and S. griseus IFO13350 and identified four new CHEs from actinomycetes. The enzymic properties of the CHEs from Streptomyces sp. X9, S. avermitilis, and S. griseus including substrate specificity, sensitivity to inhibitors and optimal conditions for catalysis were similar. We identified genes for the CHEs from Streptomyces sp. X9 and S. avermitilis and the encoded predicted sequences comprised 217 and 214 amino acid residues, respectively, with 64% similarity. The CHEs from Streptomyces sp. X9 and S. avermitilis were also 54 and 57% similar, respectively, to S. lavendulae CHE, indicating that these CHEs are orthologs. Phylogenetic analysis showed that they are distantly related to the conventional lipase/esterase type CHEs from mammals, yeasts and other bacteria. The actinomycetes CHEs did not have the Gly–Xaa–Ser–Xaa–Gly sequence that is conserved in the lipase/esterase family. A database search showed that orthologs of this type of CHE were restricted to actinomycetes. These findings imply that the actinomycetes CHEs constitute a novel family of cholesterol esterases.

Keywords: Abbreviations; pCMB; p; -chloromercuribenzoate; CHE; cholesterol esterase; CL; cholesteryl linoleate; CP; cholesteryl palmitate; DTT; dithiothreitol; βME; β-mercaptoethanol; pNP; p; -nitrophenyl; PMSF; phenylmethylsulfonyl fluoride; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresisCholesterol esterase; Cholesterol ester; Cholesterol; Lipase; Streptomyces

The pH dependence of the activity of dehaloperoxidase from Amphitrite ornata by Stefan Franzen; Lauren B. Gilvey; Jennifer L. Belyea (pp. 121-130).

Dehaloperoxidase (DHP) from the terebellid polychaete, Amphitrite ornata, is the first hemoglobin that has peroxidase activity as part of its native function. The substrate 2,4,6-tribromophenol (TBP) is oxidatively debrominated by DHP to form 2,6-dibromoquinone (DBQ) in a two-electron process. There is a well-defined internal binding site for TBP above the heme, a feature not observed in other hemoglobins or peroxidases. A study of the pH dependence of the activity of DHP reveals a substantial difference in mechanism. From direct observation of the Soret band of the heme it is shown that the p K_a for heme activation in protein DHP is 6.5. Below this pH the heme absorbance decreases in the presence of H₂O₂ with or without addition of substrate. The low pH data are consistent with significant heme degradation. Above pH 6.5 addition of H₂O₂ causes the heme to shift rapidly to a compound II spectrum and then slowly to an unidentified intermediate with an absorbance of 410 nm. However, the p K_a of the substrate TBP is 6.8 and the greatest enzyme activity is observed above the p K_a of TBP under conditions where the substrate is a phenolate anion (TPBO⁻). Although the mechanisms may differ, the data show that both neutral TBP and anionic TPBO⁻ are converted to the quinone product. The mechanistic implications of the pH dependence are discussed by comparison other known peroxidases, which oxidize substrates at the heme edge.

Keywords: Abbreviations; CcP; cytochrome; c; peroxidase; DBQ; 2,6 dibromoquinone; DCQ; 2,6 dichloroquinone; DHP; dehaloperoxidase; HRP; horseradish peroxidase; LiP; lignin peroxidase; Mb; myoglobin; P450cam; cytochrome P450 camphor; SVD; singular value decomposition; SWMb; Sperm Whale myoglobin; TBP; 2,4,6 tribromophenol; TCP; 2,4,6 trichlorophenol; 24-DCP; 2,4-dichlorophenolPeroxidase; Fourier-transform infrared spectroscopy; X-ray crystallography; Enzyme kinetics

The stress response protein Hsp12p increases the flexibility of the yeast Saccharomyces cerevisiae cell wall by Robert J. Karreman; Etienne Dague; Fabien Gaboriaud; Fabienne Quilès; Jerome F.L. Duval; George G. Lindsey (pp. 131-137).

The yeast S. cerevisiae cell wall comprising a 10 nm thick layer of polysaccharides, predominantly β(1,3)-glucan and proteins, is the interface between the cell and the neighbouring environment. As such it is not a static entity but rather one that is dynamically remodelled in response to changes in the environmental conditions. We have recently proposed from studies using yeast cells lacking the gene encoding Hsp12p ( Δhsp12 yeast) and from incorporation of Hsp12p into agarose, used as a model system for the β-glucan layer of the cell wall, that the hydrophilic stress response cell wall protein Hsp12p acts as a cell wall plasticizer. In this report we have used force spectroscopy to confirm that Δhsp12 yeast are indeed less flexible than the wild type strain. The spring constant of the cell wall of Δhsp12 yeast, k_cw was determined to be 72±3 mN m−¹ as compared to 17±5 mN m−¹ obtained for the wild type strain. A similar result was found on the basis of a quantitative analysis of the electrophoretic mobilities measured for the two yeast strains. Those indicated that the hydrodynamic permeability quantified through the softness parameter of the external layer of Δhsp12 cells was smaller than the one of wild type cells. We proposed from surface infrared spectroscopy measurements that yeast compensate for the lack of Hsp12p by reducing the carbohydrate/proteins ratio of the cell wall or increasing the cell wall chitin content.

Keywords: Abbreviations; AFM; atomic force microscopy; HSP; heat shock protein; LEA; late embryogenic abundant; YEPD; yeast extract peptone dextrose; PEI; polyethyleneimine; ATR; Attenuated Total Reflection; FTIR; Fourier Transform infrared; DTGS; deuterated triglycine sulphate Saccharomyces cerevisiae; Cell wall flexibility; Force spectroscopy; Infrared spectroscopy; Electrophoresis; Soft particle analysis; AFM

Hydrodynamically coupled water in surface adsorbed amino acids as a tool to study hydrated peptides by Muthuselvi Lakshmanan; Aruna Dhathathreyan (pp. 138-145).

The influence of different amino acid residues on properties of a protein surface is of great interest and importance. Hydrodynamically coupled water in the amino acids has the potential to be used as a tool to study surface properties of proteins. The contribution of this coupled water fraction in design of a hydropathy scale in surface adsorbed amino acid films on solid using quartz crystal microbalance is presented in this work. This scale compares well with the hydropathy scale of Guy reported in the literature and can be correlated with the solid/liquid interfacial tension and work of adhesion of the adsorbed amino acid films. Using Graphical Representation and Analysis of Surface Properties (GRASP) the free energy of transfer from Octanol to water for the amino acids has been estimated and shows approximately an inverse relationship with the coupled water fraction. This scale has been applied in a benchmark test for a native Laminin peptide YIGSR and its mutated sequences (with mutations carried out at ‘Y and ‘R’ positions). The experimentally measured coupled water fractions seem to compare well with that obtained from the present scale assuming the total solvent fraction to be a linear function of the amino acids in the sequence. A survey of the protein data bank showed that sets of sequences based on this scale occur in membrane insertion domain or in trans-membrane proteins suggesting that the scale is suitable to study structure–function correlation in proteins.

Keywords: Hydrodynamically coupled water; Amino acids; Laminin; Hydropathy scale; Solvent fraction

Characterization of protein aggregation: The case of a therapeutic immunoglobulin by Barthélemy Demeule; M. Jayne Lawrence; Alex F. Drake; Robert Gurny; Tudor Arvinte (pp. 146-153).

In this paper, a therapeutic immunoglobulin (Antibody A) has been characterized in two solutions: (1) 0.1% acetic acid containing 50 mM magnesium chloride, a solution in which the immunoglobulin is stable, and (2) 10 mM sodium phosphate buffer pH ∼7. The protein solutions were characterized by microscopy, asymmetrical flow field-flow fractionation (FFF), light scattering, circular dichroism, fluorescence and fluorescence lifetime spectroscopy. The results show that Antibody A dissolved in 0.1% acetic acid containing 50 mM magnesium chloride exists as 88% monomer, 2% low molecular weight aggregates and 10% high molecular weight aggregates (>1 million Dalton). In phosphate buffer, Antibody A formed micrometre-sized aggregates that were best characterized by fluorescence microscopy. The aggregation of Antibody A in phosphate buffer was shown to be concomitant with conformational changes in amino acid residue side chains. The aggregates formed in phosphate buffer were easily disrupted during FFF analysis, indicating that they are formed by weak interactions. The combination of microscopy, asymmetrical flow field-flow fractionation (FFF) and spectroscopy allowed a reliable assessment of protein self association and aggregation.

Keywords: Abbreviations; CD; circular dichroism; FFF; asymmetrical flow field-flow fractionation; RMS; root mean square; TCSPC; time-correlated single-photon counting; IgG; immunoglobulin GImmunoglobulin; Aggregation; Characterization; Biopharmaceutical; Conformation

Prion infection-impaired functional blocks identified by proteomics enlighten the targets and the curing pathways of an anti-prion drug by J.-F. Chich; B. Schaeffer; A.-P. Bouin; F. Mouthon; V. Labas; C. Larramendy; J.-P. Deslys; J. Grosclaude (pp. 154-167).

Prion-induced neurodegeneration results from multiple cellular alterations among which the accumulation of a modified form of the host protein PrP is but a hallmark. Drug treatments need understanding of underlying mechanisms. Proteomics allows getting a comprehensive view of perturbations leading to neuronal death. Heparan sulfate mimetics has proved to be efficient to clear scrapie protein in cultured cells and in animals. To investigate the mechanisms of drug attack, protein profiles of the neuronal cell line GT1 and its chronically Chandler strain infected counterpart were compared, either in steady state cultures or after a 4-day drug treatment. Differentially expressed proteins were associated into functional blocks relevant to neurodegenerative diseases. Protein structure repair and modification, proteolysis, cell shape and energy/oxidation players were affected by infection, in agreement with prion biology. Unexpectedly, novel affected blocks related to translation, nucleus structure and DNA replication were unravelled displaying commonalities with proliferative processes. The drug had a double action in infected cells by reversing protein levels back to normal in some blocks and by heightening survival functions in others. This study emphasizes the interest of a proteomic approach to unravel novel networks involved in prion infection and curing.

Keywords: Proteomics; Prion; Heparan sulfate mimetics; Neuronal cell line

Corrigendum to “Reversible resolution of flavin and pterin cofactors of His-tagged Escherichia coli DNA photolyase” [Biochim. Biophys. Acta 1764 (2006) 1454–1461] by Lei Xu; Dongfang Zhang; Wanmeng Mu; Willem J.H. van Berkel; Zhaofeng Luo (pp. 168-169).

Acknowledgement (pp. 170-173).

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BBA - Proteins and Proteomics (v.1774, #1)