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BBA - Proteins and Proteomics (v.1804, #4)

Editorial Board (pp. i).

Quantitative mass spectrometry of diabetic kidney tubules identifies GRAP as a novel regulator of TGF-β signaling by Timothy D. Cummins; Michelle T. Barati; Susan C. Coventry; Sarah A. Salyer; Jon B. Klein; David W. Powell (pp. 653-661).

The aim of this study was to define novel mediators of tubule injury in diabetic kidney disease. For this, we used state-of-the-art proteomic methods combined with a label-free quantitative strategy to define protein expression differences in kidney tubules from transgenic OVE26 type 1 diabetic and control mice. The analysis was performed with diabetic samples that displayed a pro-fibrotic phenotype. We have identified 476 differentially expressed proteins. Bioinformatic analysis indicated several clusters of regulated proteins in relevant functional groups such as TGF-β signaling, tight junction maintenance, oxidative stress, and glucose metabolism. Mass spectrometry detected expression changes of four physiologically relevant proteins were confirmed by immunoblot analysis. Of these, the Grb2-related adaptor protein (GRAP) was up-regulated in kidney tubules from diabetic mice and fibrotic kidneys from diabetic patients, and subsequently confirmed as a novel component of TGF-β signaling in cultured human renal tubule cells. Thus, indicating a potential novel role for GRAP in TGF-β-induced tubule injury in diabetic kidney disease. Although we targeted a specific disease, this approach offers a robust, high-sensitivity methodology that can be applied to the discovery of novel mediators for any experimental or disease condition.

Keywords: Proteomic; Mass spectrometry; TGFβ signaling; Fibrosis; Type 1 diabetes

Investigation of the role of conserved residues Ser13, Asn48 and Pro49 in the catalytic mechanism of the tau class glutathione transferase from Glycine max by Irene Axarli; Christiana Georgiadou; Prathusha Dhavala; Anastassios C. Papageorgiou; Nikolaos E. Labrou (pp. 662-667).

Plant glutathione transferases (GSTs) play a key role in the metabolism of various xenobiotics. In this report, the catalytic mechanism of the tau class GSTU4-4 isoenzyme from Glycine max ( GmGSTU4-4) was investigated by site-directed mutagenesis and steady-state kinetic analysis. The catalytic properties of the wild-type enzyme and three mutants of strictly conserved residues (Ser13Ala, Asn48Ala and Pro49Ala) were studied in 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction. The results showed that the mutations significantly affect substrate binding and specificity. The effect of Ser13Ala mutation on the catalytic efficiency of the enzyme could be explained by assuming the direct involvement of Ser13 to the reaction chemistry and the correct positioning of GSH and CDNB in the ternary catalytic complex. Asn48 and Pro49 were found to have a direct role on the structural integrity of the GSH-binding site (G-site). Moreover, mutation of Asn48 and Pro49 residues may bring about secondary effects altering the thermal stability and the catalytic activity ( k_cat) of the enzyme without affecting the nature of the rate-limiting step of the catalytic reaction.

Keywords: Abbreviations; CDNB; 1-chloro-2,4-dinitrobenzene; GSH; glutathione; GST; glutathione-S-transferase; G-site; GSH binding site; H-site; hydrophobic binding site; S; -(p-nitrobenzyl)-glutathione; Nb-GSHHerbicide detoxification; Site-directed mutagenesis; Kinetic mechanism, Tau class glutathione transferase

Transglycosylation reaction catalyzed by a class V chitinase from cycad, Cycas revoluta: A study involving site-directed mutagenesis, HPLC, and real-time ESI-MS by Toki Taira; Maho Fujiwara; Nicole Dennhart; Hiroko Hayashi; Shoko Onaga; Takayuki Ohnuma; Thomas Letzel; Shohei Sakuda; Tamo Fukamizo (pp. 668-675).

Class V chitinase from cycad, Cycas revoluta, (CrChi-A) is the first plant chitinase that has been found to possess transglycosylation activity. To identify the structural determinants that bring about transglycosylation activity, we mutated two aromatic residues, Phe166 and Trp197, which are likely located in the acceptor binding site, and the mutated enzymes (F166A, W197A) were characterized. When the time-courses of the enzymatic reaction toward chitin oligosaccharides were monitored by HPLC, the specific activity was decreased to about 5–10% of that of the wild type and the amounts of transglycosylation products were significantly reduced by the individual mutations. From comparison between the reaction time-courses obtained by HPLC and real-time ESI-MS, we found that the transglycosylation reaction takes place under the conditions used for HPLC but not under the ESI-MS conditions. The higher substrate concentration (5 mM) used for the HPLC determination is likely to bring about chitinase-catalyzed transglycosylation. Kinetic analysis of the time-courses obtained by HPLC indicated that the sugar residue affinity of +1 subsite was strongly reduced in both mutated enzymes, as compared with that of the wild type. The IC₅₀ value for the inhibitor allosamidin determined by real-time ESI-MS was not significantly affected by the individual mutations, indicating that the state of the allosamidin binding site (from −3 to −1 subsites) was not changed in the mutated enzymes. We concluded that the aromatic side chains of Phe166 and Trp197 in CrChi-A participate in the transglycosylation acceptor binding, thus controlling the transglycosylation activity of the enzyme.

Keywords: Abbreviations; GlcNAc; 2-acetamido-2-deoxy-; d; -glucopyranose; (GlcNAc); _n; β-1, 4-linked oligosaccharide of GlcNAc with a polymerization degree of; n; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; HPLC; high performance liquid chromatography. ESI-MS, electrosplay ionization mass spectrometryPlant chitinase; Oligosaccharide hydrolysis; HPLC; Real-time ESI-MS; Transglycosylation

Quantitative proteomic analysis of ribosomal protein L35b mutant of Saccharomyces cerevisiae by Yong Bhum Song; Min A Jhun; Taesung Park; Won-Ki Huh (pp. 676-683).

Recent studies have revealed that in higher eukaryotes, several ribosomal proteins are involved in some pathological events or developmental defects, indicating that ribosomal proteins perform unconventional functions other than protein biosynthesis. To obtain an insight into the novel roles of ribosomal proteins, we aimed to analyze the changes in proteome expression in ribosomal protein mutants by using Saccharomyces cerevisiae as a model system. We introduced the rpl35bΔ mutation into the 4159 green fluorescent protein (GFP)-tagged yeast strains by using the synthetic genetic array (SGA) method, and performed quantitative proteomic analysis by using a multilabel microplate reader and flow cytometer. We identified 22 upregulated and 20 downregulated proteins in the rpl35bΔ mutant. These proteins were primarily classified into the Gene Ontology (GO) categories of cellular biosynthetic process, translation, protein or nucleotide metabolic process, cell wall organization and biogenesis, and hyperosmotic response. We also investigated the correlation between the mRNA and protein levels of the identified proteins. Our results show that a ribosomal protein mutation can lead to perturbation in the expression of several proteins, including some other ribosomal proteins. Furthermore, our approach of combining a library of GFP-tagged yeast strains and the SGA method provides an effective and highly sensitive method for dynamic analysis of the effects of various mutations on proteome expression.

Keywords: Abbreviations; GFP; green fluorescent protein; SGA; synthetic genetic array; GO; Gene Ontology; 2-DE; two-dimensional gel electrophoresis; SC; synthetic complete; TAP; tandem affinity purification; PBS; phosphate-buffered saline; OD; optical density; RT; reverse transcription; SBDS; Shwachman–Bodian–Diamond syndrome proteinRibosomal protein; Saccharomyces cerevisiae; Green fluorescent protein; Synthetic genetic array; Proteome

The catalytic efficiency ( k_cat/ K_m) of the class A β-lactamase Toho-1 correlates with the thermal stability of its catalytic intermediate analog by Yasushi Nitanai; Tatsuro Shimamura; Takuro Uchiyama; Yoshikazu Ishii; Michiyo Takehira; Katsuhide Yutani; Hiroshi Matsuzawa; Masashi Miyano (pp. 684-691).

The extended-spectrum β-lactamases are associated with antibiotic resistance. Toho-1 R274N/R276N, a Class A β-lactamase of CTX-M-type, efficiently hydrolyzes first generationcephalosporins (for example, cephalothin), in addition to cefotaxime, a third generation cephalosporin. However, this enzyme only marginally hydrolyzes the third generation cephalosporin ceftazidime, and the monobactam aztreonam. The deacylation defectiveness of the mutant Toho-1 E166A/R274N/R276N, which lacks the deacylation activity, results in the accumulation of the complex of an acylated-enzyme intermediate analog. For drug design, it would be useful if a quantitative prediction of a catalytic property were available without the need of enzymatic measurements. Therefore, we examined whether there is a correlation between the thermal stability of a catalytic intermediate (analog) and its kinetic parameters. First we measured the hydrolytic kinetics of the 14 species of β-lactam antibiotics by Toho-1 R274N/R276N, and also measured the thermal stability of the accumulated acyl-intermediates of Toho-1 E166A/R274N/R276 by differential scanning calorimetry. Here we report the correlation of these parameters. The logarithm of the catalytic efficiency for Toho-1 R274N/R276N, log( k_cat/ K_m) exhibited the best linear correlation with T_m, which is the heat-denaturation temperature midpoint of the corresponding acylated complex of Toho-1 E166A/R274N/R276N. The correlation coefficient was 0.947, indicating that a relationship exists between the kinetic parameters and the stability of the intermediates. The results demonstrate a new method for investigating the catalytic properties of enzymes against any substrates, and a new approach to designing enzymes.

Keywords: CTX-M-type β-lactamase; Toho-1; Acyl-intermediate; Thermal stability; Differential scanning calorimetry; β-lactam antibiotics

Extensive association of HuR with hnRNP proteins within immunoselected hnRNP and mRNP complexes by Christina Papadopoulou; Meropi Patrinou-Georgoula; Apostolia Guialis (pp. 692-703).

Regulated gene expression at the post-transcriptional level in higher eukaryotes is based on a network of interactions among RNA-binding proteins (RBPs) operating within multifactorial ribonucleoprotein (RNP) complexes, notably heterogeneous nuclear ribonucleoprotein (hnRNP) and mRNP complexes. We are interested in interactions involving hnRNP proteins participating in several steps of mRNA processing (mainly pre-mRNA splicing) and HuR with an established role in stability/translation of associated mRNAs. hnRNP and HuR proteins have a major nucleoplasmic localization and ability to shuttle between nucleus and cytoplasm. We report here on interactions between hnRNP and HuR proteins that were identified in the context of isolated hnRNP and mRNP complexes. This was done by the application of immunoprecipitation and pull-down assays on different sub-cellular fractions prepared from cells of human and mouse origin, as well as in vivo localization studies. A range of specific associations of HuR with the shuttling hnRNP A1 and A3 and the non-shuttling hnRNP C1/C2 was identified and ascribed discrete properties with respect to stability to RNase A and increasing salt, as well as to cellular distribution. The likelihood of a biological relevance of these associations was tested under heat shock conditions in growing cells, which appeared to affect both the sub-nuclear distribution and interaction of HuR with hnRNPs. The establishment of an extensive association of HuR with hnRNP components of nuclear hnRNP/mRNP and cytoplasmic mRNP complexes supports its broader participation in mRNA processing events than initially anticipated.

Keywords: Abbreviations; hnRNP; heterogeneous nuclear ribonucleoprotein; snRNP; small nuclear ribonucleoprotein; RBPs; RNA binding proteins; TIA-1; T-cell internal antigen-1; TIAR; TIA-1-related protein; RRM; RNA recognition motif; RBD; RNA binding domain; KH domain; hnRNP K homology domain; RGG domain; Arg-Gly-Gly domain; 3′ UTR; 3′ untranslated region; ARE; AU-rich element; AUF1; AU-binding factor 1; ELAV; embryonic lethal abnormal vision protein; FRET; fluorescence resonance energy transfer; 2-D; two dimensional; SAF-A; scaffold attachment factor A; NEPHGE; non-equilibrium pH gradient electrophoresis; SDS-PAGE; SDS-polyacrylamide gel electrophoresisRNA-binding protein; RNA-protein interaction; mRNA stability/translation; Heat shock; ARE-binding protein

Solvent-assisted slow conversion of a dithiazole derivative produces a competitive inhibitor of peptide deformylase by Alexander K. Berg; Qingfeng Yu; Steven Y. Qian; Manas K. Haldar; D.K. Srivastava (pp. 704-713).

Due to its potential as an antibiotic target, E. coli peptide deformylase (PDF_Ec) serves as a model enzyme system for inhibitor design. While investigating the structural–functional and inhibitory features of this enzyme, we unexpectedly discovered that 2-amino-5-mercapto-1,3,4-thiadiazole (AMT) served as a slow-binding inhibitor of PDF_Ec when the above compound was dissolved only in dimethylformamide (DMF), but not in any other solvent, and allowed to age. The time dependent inhibitory potency of the DMF-dissolved AMT was correlated with the broadening of the inhibitor's 295 nm spectral band toward the visible region, concomitant with the increase in the mass of the parent compound by about 2-fold. These data led to the suggestion that DMF facilitated the slow dimerization of AMT (via the formation of a disulfide bond), and that the dimeric form of AMT served as an inhibitor for PDF_Ec. The latter is not caused by the simple oxidation of sulfhydryl groups by oxidizing agents such as H₂O₂. Newly synthesized dimeric/dithiolated form of AMT (“bis-AMT”) exhibited similar spectral and inhibitory features as given by the parent compound when incubated with DMF. The computer graphic modeling data revealed that bis-AMT could be reliably accommodated within the active site pocket of PDF_Ec, and the above enzyme–ligand interaction involves coordination with the enzyme resident Ni²⁺ cofactor. The mechanism of the DMF-assisted activation of AMT (generating bis-AMT), the overall microscopic pathway for the slow-binding inhibition of PDF_Ec by bis-AMT, and the potential of bis-AMT to serve as a new class of antibiotic agent are presented.

Keywords: Abbreviations; PDF; peptide deformylase; Ec; E. coli; AMT; 2-amino-5-mercapto-1,3,4-thiadiazole; DMF; N,N′-dimethylformamide; NME; N-terminal methionine excision; ^F; Met; formyl-methionine; DMSO; dimethylsulfoxide; BSA; bovine serum albumin; HOAc; acetic acid; ESI; electrospray ionization; Sa; S. aureusPeptide deformylase; Inhibitor; Slow inhibition; Thiadiazole

Chemical modification of lysine residues in lysozyme may dramatically influence its amyloid fibrillation by Dina Morshedi; Azadeh Ebrahim-Habibi; Ali Akbar Moosavi-Movahedi; Mohsen Nemat-Gorgani (pp. 714-722).

Studies on the aggregation of mutant proteins have provided new insights into the genetics of amyloid diseases and the role of the net charge of the protein on the rate, extent, and type of aggregate formation. In the present work, hen egg white lysozyme (HEWL) was employed as the model protein. Acetylation and (separately) citraconylation were employed to neutralize the charge on lysine residues. Acetylation of the lysine residues promoted amyloid formation, resulting in more pronounced fibrils and a dramatic decline in the nucleation time. In contrast, citraconylation produced the opposite effect. In both cases, native secondary and tertiary structures appeared to be retained. Studies on the effect of pH on aggregation suggested greater possibilities for amorphous aggregate formation rather than fibrillation at pH values closer to neutrality, in which the protein is known to take up a conformation more similar to its native form. This is in accord with reports in the literature suggesting that formation of amorphous aggregates is more favored under relatively more native conditions. pH 5 provided a critical environment in which a mixture of amorphous and fibrillar structures were observed. Use of Tango and Aggrescan software which describe aggregation tendencies of different parts of a protein structure suggested critical importance of some of the lysine residues in the aggregation process. Results are discussed in terms of the importance of the net charge in control of protein–protein interactions leading to aggregate formation and possible specific roles of lysine residues 96 and 97.

Keywords: Abbreviations; HEWL; hen egg white lysozyme; ThT; thioflavin T; ANS; 8-anillino-1-naphthalene-sulfonic acid; TEM; transmission electron microscopy; Ac-HEWL; acetylated HEWL; Ac-Lys; acetylated lysine; Cit-HEWL; citraconylated-HEWL; Cit-Lys; citraconylated-lysine; 4-MU-β-GlcNAc3; 4-Nitrophenyl β-; d; -; N; ,; N; ′,; N; ′′-triacetylchitotriose; ptm; post-translational modification; I3C; indole-3-carbinolAmyloid fibrillation; Amorphous aggregate; Lysozyme; Chemical modification; Acetylation; Citraconylation

Enhanced fibrinolysis by proteolysed coagulation factor Xa by Kimberley Talbot; Scott C. Meixner; Edward L.G. Pryzdial (pp. 723-730).

We previously showed that coagulation factor Xa (FXa) enhances activation of the fibrinolysis zymogen plasminogen to plasmin by tissue plasminogen activator (tPA). Implying that proteolytic modulation occurs in situ, intact FXa (FXaα) must be sequentially cleaved by plasmin or autoproteolysis, producing FXaβ and Xa33/13, which acquire necessary plasminogen binding sites. The implicit function of Xa33/13 in plasmin generation has not been demonstrated, nor has FXaα/β or Xa33/13 been studied in clot lysis experiments. We now report that purified Xa33/13 increases tPA-dependent plasmin generation by at least 10-fold. Western blots confirmed that in situ conversion of FXaα/β to Xa33/13 correlated to enhanced plasmin generation. Chemical modification of the FXaα active site resulted in the proteolytic generation of a product distinct from Xa33/13 and inhibited the enhancement of plasminogen activation. Identical modification of Xa33/13 had no effect on tPA cofactor function. Due to its overwhelming concentration in the clot, fibrin is the accepted tPA cofactor. Nevertheless, at the functional level of tPA that circulates in plasma, FXaα/β or Xa33/13 greatly reduced purified fibrin lysis times by as much as 7-fold. This effect was attenuated at high levels of tPA, suggesting a role when intrinsic plasmin generation is relatively low. FXaα/β or Xa33/13 did not alter the apparent size of fibrin degradation products, but accelerated the initial cleavage of fibrin to fragment X, which is known to optimize the tPA cofactor activity of fibrin. Thus, coagulation FXaα undergoes proteolytic modulation to enhance fibrinolysis, possibly by priming the tPA cofactor function of fibrin.

Keywords: Fibrinolysis; Coagulation; Factor Xa; Tissue plasminogen activator; Plasminogen; Plasmin

MMP-12 catalytic domain recognizes and cleaves at multiple sites in human skin collagen type I and type III by Samuel Taddese; Michael C. Jung; Christian Ihling; Andrea Heinz; Reinhard H.H. Neubert; Christian E.H. Schmelzer (pp. 731-739).

Collagens of either soft connective or mineralized tissues are subject to continuous remodeling and turnover. Undesired cleavage can be the result of an imbalance between proteases and their inhibitors. Owing to their superhelical structure, collagens are resistant to many proteases and matrix metalloproteinases (MMPs) are required to initiate further degradation by other enzymes. Several MMPs are known to degrade collagens, but the action of MMP-12 has not yet been studied in detail. In this work, the potential of MMP-12 in recognizing sites in human skin collagen types I and III has been investigated. The catalytic domain of MMP-12 binds to the triple helix and cleaves the typical sites -Gly₇₇₅-Leu₇₇₆- in α-2 type I collagen and -Gly₇₇₅-Ile₇₇₆- in α-1 type I and type III collagens and at multiple other sites in both collagen types. Moreover, it was observed that the region around these typical sites contains comparatively less prolines, of which some have been proven to be only partially hydroxylated. This is of relevance since partial hydroxylation in the vicinity of a potential scissile bond may have a local effect on the conformational thermodynamics with probable consequences on the collagenolysis process. Taken together, the results of the present work confirm that the catalytic domain of MMP-12 alone binds and degrades collagens I and III.

Keywords: Collagenolysis; Hydroxylation; Mass spectrometry; Macrophage elastase; Cleavage site specificity; Extracellular matrix

Non-linear effects of macromolecular crowding on enzymatic activity of multi-copper oxidase by Irina Pozdnyakova; Pernilla Wittung-Stafshede (pp. 740-744).

Enzymes catalyze biochemical reactions in highly crowded environments where the amount of macromolecules may occupy up to 40% of the volume. Here we report how cell-like conditions tune catalytic parameters for the monomeric multi-copper oxidase, Saccharomyces cerevisiae Fet3p, in vitro. At low amounts of crowding agent, we detect increases in both of K_M (weaker substrate binding) and k_cat (improved catalytic efficiency), whereas at higher crowding levels, both parameters were reduced. Presence of crowding agents does not affect Fet3p structural content but increases thermal resistance. The observations are compatible with ordering of a non-optimal substrate-binding site and restricted internal dynamics as a result of excluded volume effects making the protein less structurally ‘strained’.

Keywords: Abbreviations; CD; circular dichroism; MCO; multi-copper oxidaseEnzyme activity; Macromolecular crowding; Circular dichroism; Thermal stability; Crowding agent

Proteomic analysis of endogenous conjugated linoleic acid biosynthesis in lactating rats and mouse mammary gland epithelia cells (HC11) by Yong-Cheng Jin; Hong-Gu Lee; Cheng-Xiong Xu; Jeng-A Han; Seong-Ho Choi; Man-Kang Song; Young-Jun Kim; Ki-Beom Lee; Seon-Ku Kim; Han-Seok Kang; Byung-Wook Cho; Teak-Soon Shin; Yun-Jaie Choi (pp. 745-751).

This study was conducted to investigate the amount of CLA synthesized endogenously by rat mammary tissues in response to TVA (a precursor for cis-9, trans-11 CLA endogenous synthesis) treatment as well as the differences in the protein expression of genes encoding the biosynthesis of CLA in rat mammary tissue and mouse mammary gland epithelia cells (HC11). Treatment with TVA resulted in improved CLA productivity. Furthermore, 2-DE revealed two spots in samples of mammary tissues and one spot in samples of mammary gland epithelia cells (HC11) that were consistently altered in the TVA treatment groups when compared with the control group (non-fatty acid). The mRNA expression patterns of three of the proteins (PDI, PRDX2, LAMR1), as measured by real-time PCR, were similar to the pattern of protein abundance. In addition, the expression of SCD mRNA in the mammary tissue of rats and HC11 cell treated with TVA was higher than in the control group. Our results suggest that the identified proteins may be related to CLA biosynthesis in mammary tissue.

Keywords: Abbreviations; CLA; conjugated linoleic acid; TVA; trans; vaccenic acid; PDI; protein disulfide-isomerase precursor; PRDX2; peroxiredoxin-2; LAMR1; laminin receptor 1; SCD; stearoyl-CoA desaturase; FAS; fatty acid synthase; LPL; lipoprotein lipaseConjugated linoleic acid; Trans; vaccenic acid; Two-dimensional electrophoresis; Real-time PCR; Stearoyl-CoA desaturase

¹³C isotope effect on the reaction catalyzed by prephenate dehydratase by Jeremy Van Vleet; Andreas Kleeb; Peter Kast; Donald Hilvert; W.W. Cleland (pp. 752-754).

The¹³C isotope effect for the conversion of prephenate to phenylpyruvate by the enzyme prephenate dehydratase from Methanocaldococcus jannaschii is 1.0334±0.0006. The size of this isotope effect suggests that the reaction is concerted. From the X-ray structure of a related enzyme, it appears that the only residue capable of acting as the general acid needed for removal of the hydroxyl group is threonine-172, which is contained in a conserved TRF motif. The more favorable entropy of activation for the enzyme-catalyzed process (25 eu larger than for the acid-catalyzed reaction) has been explained by a preorganized microenvironment that obviates the need for extensive solvent reorganization. This is consistent with forced planarity of the ring and side chain, which would place the leaving carboxyl and hydroxyl out of plane. Such distortion of the substrate may be a major contributor to catalysis.

Keywords: Abbreviations; PDT; prephenate dehydratase; MjPDT; PDT from; Methanocaldococcus jannaschii; KIE; kinetic isotope effect; IRMS; isotope ratio mass spectrometryPrephenate dehydratase; ¹³; C Isotope effect; Transition state structure

C-terminal domain of the Uup ATP-binding cassette ATPase is an essential folding domain that binds to DNA by Monica Y. Burgos Zepeda; Kevin Alessandri; Dorothée Murat; Chahrazade El Amri; Elie Dassa (pp. 755-761).

The Uup protein belongs to a subfamily of soluble ATP-binding cassette (ABC) ATPases that have been implicated in several processes different from transmembrane transport of molecules, such as transposon precise excision. We have demonstrated previously that Escherichia coli Uup is able to bind DNA. DNA binding capacity is lowered in a truncated Uup protein lacking its C-terminal domain (CTD), suggesting a contribution of CTD to DNA binding. In the present study, we characterize the role of CTD in the function of Uup, on its overall stability and in DNA binding. To this end, we expressed and purified isolated CTD and we investigated the structural and functional role of this domain. The results underline that CTD is essential for the function of Uup, is stable and able to fold up autonomously. We compared the DNA binding activities of three versions of the protein (Uup, UupΔCTD and CTD) by an electrophoretic mobility shift assay. CTD is able to bind DNA although less efficiently than intact Uup and UupΔCTD. These observations suggest that CTD is an essential domain that contributes directly to the DNA binding ability of Uup.

Keywords: Soluble ABC ATPase; Transposon precise excision; C-terminal domain; DNA binding; Oligomerization; Folding

Photochemical processes observed during the reaction of superoxide reductase from Desulfoarculus baarsii with superoxide by Florence Bonnot; Chantal Houée-Levin; Vincent Favaudon; Vincent Nivière (pp. 762-767).

Superoxide reductase SOR is an enzyme involved in superoxide detoxification in some microorganisms. Its active site consists of a non-heme ferrous center in an unusual [Fe(NHis)₄ (SCys)₁] square pyramidal pentacoordination that efficiently reduces superoxide into hydrogen peroxide. In previous works, the reaction mechanism of the SOR from Desulfoarculus baarsii enzyme, studied by pulse radiolysis, was shown to involve the formation of two reaction intermediates T1 and T2. However, the absorption spectrum of T2 was reported with an unusual sharp band at 625 nm, very different from that reported for other SORs. In this work, we show that the sharp band at 625 nm observed by pulse radiolysis reflects the presence of photochemical processes that occurs at the level of the transient species formed during the reaction of SOR with superoxide. These processes do not change the stoichiometry of the global reaction. These data highlight remarkable photochemical properties for these reaction intermediates, not previously suspected for iron-peroxide species formed in the SOR active site. We have reinvestigated the reaction mechanism of the SOR from D. baarsii by pulse radiolysis in the absence of these photochemical processes. The T1 and T2 intermediates now appear to have absorption spectra similar to those reported for the Archaeoglobus fulgidus SOR enzymes. Although for some enzymes of the family only one transient was reported, on the whole, the reaction mechanisms of the different SORs studied so far seem very similar, which is in agreement with the strong sequence and structure homologies of their active sites.

Keywords: Abbreviations; SOR; superoxide reductase; SOD; superoxide dismutaseSuperoxide reductase; Pulse radiolysis; Reaction intermediates; Photochemical processes; Desulfoarculus baarsii

Cross-linking with bifunctional reagents and its application to the study of the molecular symmetry and the arrangement of subunits in hexameric protein oligomers by Abdussalam Azem; Yossi Tsfadia; Omar Hajouj; Isabella Shaked; Ezra Daniel (pp. 768-780).

Cross-linking with a bifunctional reagent and subsequent SDS gel electrophoresis is a simple but effective method to study the symmetry and arrangement of subunits in oligomeric proteins. In this study, theoretical expressions for the description of cross-linking patterns were derived for protein homohexamers through extension of the method used for tetramers by Hajdu et al. (1976). The derived equations were used for the analysis of cross-linking by glutardialdehyde of four protein hexamers: beef liver glutamate dehydrogenase (GDH), jack bean urease, hemocyanin from the spiny lobster Panulirus pencillatus ( PpHc), Escherichia coli glutamate decarboxylase (GDC) and for analysis of published data on the cross-linking of hexameric E. coli rho by dimethyl suberimidate. Best fit models showed that the subunits in the first four proteins are arranged according to D₃ symmetry in two layers, each subunit able to cross-link to three neighboring subunits for GDH and urease, or to four for PpHc and GDC. The findings indicate a dimer-of-trimers eclipsed arrangement of subunits for GDH and urease and a trimer-of-dimers staggered one for PpHc and GDC. In rho, the subunits are arranged according to D₃ symmetry in a trimer-of-dimers ring. The conclusions from cross-linking of GDH and GDC, PpHc and rho are consistent with results from X-ray crystal structure, those for urease with findings from electron microscopy.

Keywords: Abbreviations; GDA; glutardialdehyde; GDH; glutamate dehydrogenase; Pp; Hc; Panulirus pencillatus; hemocyanin; GDC; glutamate decarboxylase; DD; squared deviation for an individual exposure to GDA; SDD; sum of squared deviations for a set of such exposuresCross-linking; Protein hexamer; Subunit; Quaternary structure; Molecular symmetry

Dipeptidyl peptidase 9 (DPP9) from bovine testes: Identification and characterization as the short form by mass spectrometry by Véronique Dubois; Anne-Marie Lambeir; Stefaan Vandamme; Veerle Matheeussen; Yves Guisez; Scharpe Simon Scharpé; Ingrid De Meester (pp. 781-788).

The dipeptidyl peptidases (DPP) 8 and 9 belong to the DPP4 activity and/or structure homologues (DASH). Recently, a DPP9-like protein was purified from bovine testes. The aim of the present study was to prove its identity and to investigate the characteristics of this natural enzyme. We report the identification and N-terminal sequence analysis by MALDI-TOF/TOF MS, of the purified bovine enzyme as DPP9. The tryptic peptides after in-gel digestion covered 41% and 38% of the short and full-length variants of bovine DPP9, respectively. Using Asp-N digestion combined with a very recently described mass spectrometric method using DITC glass beads, the N-terminal peptide (XTGALTSERG) was isolated. It corresponds to the N-terminus of the short form of bovine DPP9. There was no evidence for glycosylation of purified bovine DPP9. The purified DPP9 was activated and stabilized by DTT. Bovine DPP9 lost its activity almost completely after alkylation with N-ethylmaleimide. Also alkylation with iodoacetamide inhibited DPP9, albeit only 70%. Other properties of bovine DPP9 are reported, including functional stability and sensitivity towards metal ions. Our results indicate that the short form of DPP9 can be isolated from bovine testes and that it behaves as a stable enzyme suitable for further functional and biochemical characterization as well as for inhibitor screening and characterization.

Keywords: Abbreviations; BSA; bovine serum albumin; DASH; DPP4 activity and/or structure homologues; DITC; p; -phenylene diisothiocyanate; DPP; dipeptidyl peptidase; DTT; dithiothreitol; FAPα; fibroblast activation protein α; MALDI; matrix assisted laser desorption ionisation; MS; mass spectrometry; ORF; open reading frame; PEP; prolyl endopeptidase; pNA; p; -nitroaniline; rDPP; recombinant human DPP; TFA; trifluoroacetic acid; TOF; time-of-flightDipeptidyl peptidase (DPP); DPP9; Bovine; Mass spectrometry (MS); N-terminal sequence analysis; Prolyl oligopeptidase family

Thermal aggregation of glycated bovine serum albumin by Philippe Rondeau; Giovanna Navarra; Francesco Cacciabaudo; Maurizio Leone; Emmanuel Bourdon; Valeria Militello (pp. 789-798).

Aggregation and glycation processes in proteins have a particular interest in medicine fields and in food technology. Serum albumins are model proteins which are able to self-assembly in aggregates and also sensitive to a non-enzymatic glycation in cases of diabetes. In this work, we firstly reported a study on the glycation and oxidation effects on the structure of bovine serum albumin (BSA). The experimental approach is based on the study of conformational changes of BSA at secondary and tertiary structures by FTIR absorption and fluorescence spectroscopy, respectively. Secondly, we analysed the thermal aggregation process on BSA glycated with different glucose concentrations. Additional information on the aggregation kinetics are obtained by light scattering measurements. The results show that glycation process affects the native structure of BSA. Then, the partial unfolding of the tertiary structure which accompanies the aggregation process is similar both in native and glycated BSA. In particular, the formation of aggregates is progressively inhibited with growing concentration of glucose incubated with BSA. These results bring new insights on how aggregation process is affected by modification of BSA induced by glycation.

Keywords: Abbreviations; AGE; advanced glycated end; ANSA; 1-anilino-8-naphthalene sulfonic acid; BSA; bovine serum albumin; DNPH; 2,4-dinitrophénylhydrazine; MES; 2-(; N; -morpholino)ethane sulfonic acid; PAGE; polyacrylamide gel electrophoresis; PBS; phosphate-buffered saline; ELISA; enzyme-linked immunosorbent assay; FTIR; Fourier transform infrared; DLS; dynamic light scatteringGlycoxidation; Albumin; Protein aggregation; FTIR spectroscopy; Light scattering; Glycation

Probing hydrogen peroxide oxidation kinetics of wild-type Synechocystis catalase-peroxidase (KatG) and selected variants by Jutta Vlasits; Furtmuller Paul G. Furtmüller; Christa Jakopitsch; Marcel Zamocky; Christian Obinger (pp. 799-805).

Catalase-peroxidases (KatGs) are unique bifunctional heme peroxidases that exhibit peroxidase and substantial catalase activities. Nevertheless, the reaction pathway of hydrogen peroxide dismutation, including the electronic structure of the redox intermediate that actually oxidizes H₂O₂, is not clearly defined. Several mutant proteins with diminished overall catalase but wild-type-like peroxidase activity have been described in the last years. However, understanding of decrease in overall catalatic activity needs discrimination between reduction and oxidation reactions of hydrogen peroxide. Here, by using sequential-mixing stopped-flow spectroscopy, we have investigated the kinetics of the transition of KatG compound I (produced by peroxoacetic acid) to its ferric state by trapping the latter as cyanide complex. Apparent bimolecular rate constants (pH 6.5, 20°C) for wild-type KatG and the variants Trp122Phe (lacks KatG-typical distal adduct), Asp152Ser (controls substrate access to the heme cavity) and Glu253Gln (channel entrance) are reported to be 1.2×10⁴M−¹s−¹, 30M−¹s−¹, 3.4×10³M−¹s−¹, and 8.6×10³M−¹s−¹, respectively. These findings are discussed with respect to steady-state kinetic data and proposed reaction mechanism(s) for KatG. Assets and drawbacks of the presented method are discussed.

Keywords: Abbreviations; KatG; catalase-peroxidase; BLC; bovine liver catalase; PAA; peroxoacetic acid; RFQ-EPR; rapid freeze-quench electron paramagnetic resonance; HS; high-spin, LS, low-spin; 5-c; five coordinated; 6-c; six coordinated; k; _on; apparent bimolecular rate constant of cyanide binding to ferric heme protein; k; ₁; ,; _app; apparent bimolecular rate constant of compound I formation; k; ₂; ,; _app; apparent bimolecular rate constant of the transition of compound I to ferric KatG; Por; porphyrin; Add; covalent Trp-Tyr-Met adductCatalase-peroxidase; Hydrogen peroxide reduction; Hydrogen peroxide oxidation; Catalase activity; Compound I; Cyanide complex; Stopped-flow spectroscopy

Divorcing folding from function: How acylation affects the membrane-perturbing properties of an antimicrobial peptide by Brian Vad; Line Aagot Thomsen; Kresten Bertelsen; Magnus Franzmann; Jan Mondrup Pedersen; Søren B. Nielsen; Thomas Vosegaard; Zuzana Valnickova; Troels Skrydstrup; Jan J. Enghild; Reinhard Wimmer; Niels Chr. Nielsen; Daniel E. Otzen (pp. 806-820).

Many small cationic peptides, which are unstructured in aqueous solution, have antimicrobial properties. These properties are assumed to be linked to their ability to permeabilize bacterial membranes, accompanied by the transition to an α-helical folding state. Here we show that there is no direct link between folding of the antimicrobial peptide Novicidin (Nc) and its membrane permeabilization. N-terminal acylation with C8–C16 alkyl chains and the inclusion of anionic lipids both increase Nc's ability to form α-helical structure in the presence of vesicles. Nevertheless, both acylation and anionic lipids reduce the extent of permeabilization of these vesicles and lead to slower permeabilization kinetics. Furthermore, acylation significantly decreases antimicrobial activity. Although acyl chains of increasing length also increase the tendency of the peptides to aggregate in solution, this cannot rationalize our results since permeabilization and antimicrobial activities are observed well below concentrations where aggregation occurs. This suggests that significant induction of α-helical structure is not a prerequisite for membrane perturbation in this class of antimicrobial peptides. Our data suggests that for Nc, induction of α-helical structure may inhibit rather than facilitate membrane disruption, and that a more peripheral interaction may be the most efficient permeabilization mechanism. Furthermore, acylation leads to a deeper embedding in the membrane, which could lead to an anti-permeabilizing “plugging” effect.

Keywords: Abbreviations; AMP; antimicrobial peptide; CD; circular dichroism; Nc; Novicidin; Nc-CX; N-terminally acylated Nc where X refers to the number of carbon atoms in the acyl chain; PRE; paramagnetic relaxation enhancementNovicidin; Membrane permeabilization; Confocal laser scanning microscopy; Circular dichroism; Liquid- and solid-state NMR; Peptide acylation

Hofmeister effects on activity and stability of alkaline phosphatase by Zhen Yang; Xiu-Ju Liu; Chao Chen; Peter J. Halling (pp. 821-828).

We have studied the effects on alkaline phosphatase of adding high concentrations (normally 1.0 M) of simple salts. It is necessary to allow for significant effects of salts on the extinction coefficient of the reaction product, and on the apparent pH of the buffer. Both activity and stability of the enzyme correlate well with the Hofmeister series in terms of the salt's kosmotropic/chaotropic properties, which are assessed by the Jones–Dole viscosity B coefficients ( B₊ for cations and B ₋ for anions). The catalytic activity or V_max/ K_m of the enzyme showed a bell-shaped relationship with the (B ₋− B₊) values of the salts present, being optimal with salts (such as NaCl, KCl, and KNO₃) where the anion and cation have similar kosmotropic/chaotropic properties. This effect is believed to be enzyme-specific and relates to the impact of both cations and anions on the enzyme's surface pH, active site, and catalytic mechanism. Anions play a more predominant role than cations in affecting enzyme stability. The rate of irreversible thermal inactivation is strongly reduced by addition of kosmotropic anions like SO₄²⁻ (half-life increased from 8 to 580 min at 60 °C). This effect is general and the mechanism probably involves the ability of the ions to affect the water solvation layer around the enzyme molecule and to interact with both the surface and internal structure of the enzyme.

Keywords: Alkaline phosphatase; Activity and stability; Hofmeister series; Kosmotropicity; Chaotropicity; Jones–Dole viscosity; B; coefficient

Modulation of advanced glycation endproduct synthesis by kynurenines in human lens proteins by Ram H. Nagaraj; Smitha Padmanabha; Maneesh Mailankot; Magdalena Staniszewska; Liew Jun Mun; Marcus A. Glomb; Mikhail D. Linetsky (pp. 829-838).

Human lens proteins (HLP) become chemically modified by kynurenines and advanced glycation end products (AGEs) during aging and cataractogenesis. We investigated the effects of kynurenines on AGE synthesis in HLP. We found that incubation with 5mM ribose or 5mM ascorbate produced significant quantities of pentosidine, and this was further enhanced in the presence of two different kynurenines (200–500µM): N-formylkynurenine (Nfk) and kynurenine (Kyn). Another related compound, 3-hydroxykynurenine (3OH-Kyn), had disparate effects; low concentrations (10–200µM) promoted pentosidine synthesis, but high concentrations (200–500µM) inhibited it. 3OH-Kyn showed similar effects on pentosidine synthesis from Amadori-enriched HLP or ribated lysine. Chelex-100 treatment of phosphate buffer reduced pentosidine synthesis from Amadori-enriched HLP by ∼90%, but it did not inhibit the stimulating effect of 3OH-Kyn and EDTA. 3OH-Kyn (100–500μM) spontaneously produced copious amounts of H₂O₂ (10–25μM), but externally added H₂O₂ had only a mild stimulating effect on pentosidine but had no effect on N^ε-carboxymethyl lysine (CML) synthesis in HLP from ribose and ascorbate. Further, human lens epithelial cells incubated with ribose and 3OH-Kyn showed higher intracellular pentosidine than cells incubated with ribose alone. CML synthesis from glycating agents was inhibited 30 to 50% by 3OH-Kyn at concentrations of 100–500μM. Argpyrimidine synthesis from 5mM methylglyoxal was slightly inhibited by all kynurenines at concentrations of 100–500μM. These results suggest that AGE synthesis in HLP is modulated by kynurenines, and such effects indicate a mode of interplay between kynurenines and carbohydrates important for AGE formation during lens aging and cataract formation.

Keywords: Abbreviations; HLP; human lens protein; Kyn; kynurenine; Nfk; N-; formyl kynurenine; 3OH-Kyn; 3OH-kynurenine; MGO; methylglyoxal; AGEs; advanced glycation end product; CML; N; ^ε; -carboxymethyl lysine; IDO; indoleamine 2, 3-dioxygenase; BSA; bovine serum albuminKynurenine; Glycation; Lens protein; Cataract

Structure and function of yeast glutaredoxin 2 depend on postranslational processing and are related to subcellular distribution by Pablo Porras; Brian McDonagh; Jose Rafael Pedrajas; Barcena J. Antonio Bárcena; C. Alicia Padilla (pp. 839-845).

We have previously shown that glutaredoxin 2 (Grx2) from Saccharomyces cerevisiae localizes at 3 different subcellular compartments, cytosol, mitochondrial matrix and outer membrane, as the result of different postranslational processing of one single gene. Having set the mechanism responsible for this remarkable phenomenon, we have now aimed at defining whether this diversity of subcellular localizations correlates with differences in structure and function of the Grx2 isoforms. We have determined the N-terminal sequence of the soluble mitochondrial matrix Grx2 by mass spectrometry and have determined the exact cleavage site by Mitochondrial Processing Peptidase (MPP). As a consequence of this cleavage, the mitochondrial matrix Grx2 isoform possesses a basic tetrapeptide extension at the N-terminus compared to the cytosolic form. A functional relationship to this structural difference is that mitochondrial Grx2 displays a markedly higher activity in the catalysis of GSSG reduction by the mitochondrial dithiol dihydrolipoamide. We have prepared Grx2 mutants affected on key residues inside the presequence to direct the protein to one single cellular compartment; either the cytosol, the mitochondrial membrane or the matrix and have analyzed their functional phenotypes. Strains expressing Grx2 only in the cytosol are equally sensitive to H₂O₂ as strains lacking the gene, whereas those expressing Grx2 exclusively in the mitochondrial matrix are more resistant. Mutations on key basic residues drastically affect the cellular fate of the protein, showing that evolutionary diversification of Grx2 structural and functional properties are strictly dependent on the sequence of the targeting signal peptide.

Keywords: Glutaredoxin; Targeting presequence; Subcellular localization; Oxidative stress; Membrane insertion; TOM20

Coupling effects of distal loops on structural stability and enzymatic activity of Escherichia coli dihydrofolate reductase revealed by deletion mutants by Yuji Horiuchi; Eiji Ohmae; Shin-ichi Tate; Kunihiko Gekko (pp. 846-855).

Residues distal from the active site in dihydrofolate reductase (DHFR) have regulatory roles in catalytic reaction and also folding stability. The couplings of the distal residues to the ones in the active site have been analyzed using site-directed mutants. To expand our understanding of the structural and functional influences of distal residue mutation, we explored the structural stability and enzymatic activity of deletion mutants. Deletion has greater structural and dynamical impacts on the corresponding part than site-directed mutation does. Thus, deletion amplifies the effects caused by distal mutations, which should make the mutual couplings among the distant residues more apparent. We focused on residues 52, 67, 121, and 145 in the four distinct loops of DHFR. All the single-residue deletion mutants showed marked reduction in stability, except for Δ52 in an αC–βC loop. Double deletion mutants showed that the loop αC–βC has nonadditive couplings with the βF–βG and βG–βH loops regarding stability. Single deletion to the loops αC–βC or βC–βD resulted in considerable activity reduction, demonstrating that the loops couple to the residues near the active site. The four loops were shown to be functionally interdependent from the double deletion experiments.

Keywords: DHFR; Deletion mutant; Enzymatic reaction; Protein folding

Rescue of F508del-CFTR by RXR motif inactivation triggers proteome modulation associated with the unfolded protein response by Patrícia Gomes-Alves; Francisco Couto; Cátia Pesquita; Ana V. Coelho; Deborah Penque (pp. 856-865).

F508del-CFTR, the most common mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, disrupts intracellular trafficking leading to cystic fibrosis (CF). The trafficking defect of F508del-CFTR can be rescued by simultaneous inactivation of its four RXR motifs (4RK). Proteins involved in the F508del-CFTR trafficking defect and/or rescue are therefore potential CF therapeutic targets. We sought to identify these proteins by investigating differential proteome modulation in BHK cells over-expressing wt-CFTR, F508del-CFTR or the revertant F508del/4RK-CFTR. By 2-dimensional electrophoresis-based proteomics and western blot approaches we demonstrated that over-expression of F508del/4RK-CFTR modulates the expression of a large number of proteins, many of which are reported interactors of CFTR and/or 14-3-3 with potential roles in CFTR trafficking. GRP78/BiP, a marker of ER stress and unfolded protein response (UPR), is up-regulated in cells over-expressing either F508del-CFTR or F598del/4RK-CFTR. However, over-expression of F508del/4RK-CFTR induces the up-regulation of many other UPR-associated proteins (e.g. GRP94, PDI, GRP75/mortalin) and, interestingly, the down-regulation of proteasome components associated with CFTR degradation, such as the proteasome activator PA28 (PSME2) and COP9 signalosome (COPS5/CSN5). Moreover, the F508del-CFTR-induced proteostasis imbalance, which involves some heat shock chaperones (e.g. HSP72/Hpa2), ER-EF-hand Ca²⁺-binding proteins (calumenin) and the proteasome activator PA28 (PSME2), tends to be ‘restored’, i.e., in BHK cells over-expressing F508del/4RK-CFTR those proteins tend to have expression levels similar to the wild-type ones. These findings indicate that a particular cellular environment orchestrated by the UPR contributes to and/or is compatible with F508del/4RK-CFTR rescue.

Keywords: Cystic fibrosis; CFTR; Unfolded protein response; Protein trafficking; RXR motif; Therapeutic target

Chaperonin-encapsulation of proteins for NMR by Shinji Tanaka; Yasushi Kawata; Gottfried Otting; Nicholas E. Dixon; Katsumi Matsuzaki; Masaru Hoshino (pp. 866-871).

A novel chaperonin-encapsulation system for NMR measurements has been designed. The single-ring variant SR398 with an ATPase deficient mutation of GroEL, also known as chaperonin, bound co-chaperonin GroES irreversibly, forming a stable cage to encapsulate a target protein. A small GroEL-binding tag made it possible to perform all steps of the encapsulation under near physiological conditions while retaining the native conformation of the target protein. About half of the SR398/GroES cages encapsulated target protein molecules. As binding only depends on the 12-residue tag sequence, this encapsulation method is applicable to a large number of proteins. Isolation of the target proteins in the molecular cage of chaperonin will allow the study of highly aggregation-prone proteins by solution NMR.

Keywords: Abbreviations; NMR; nuclear magnetic resonance; HSQC; heteronuclear single quantum coherence; SR398; single-ring mutant of GroEL with ATPase deficient mutation D398A; SBP; strongly binding peptide to GroELAggregation; NMR; Molecular chaperone; GroE; Protein encapsulation

MeuTXKβ1, a scorpion venom-derived two-domain potassium channel toxin-like peptide with cytolytic activity by Shunyi Zhu; Bin Gao; André Aumelas; del Carmen Rodriguez Maria del Carmen Rodríguez; Humberto Lanz-Mendoza; Steve Peigneur; Elia Diego-Garcia; Marie-France Martin-Eauclaire; Jan Tytgat; Lourival D. Possani (pp. 872-883).

Recent studies have demonstrated that scorpion venom contains unique two-domain peptides with the peculiarity of possessing different functions, i.e. neurotoxic and cytolytic activities. Here we report systematic characterization of a new two-domain peptide (named MeuTXKβ1) belonging to the TsTXKβ molecular subfamily from the scorpion Mesobuthus eupeus by molecular cloning, biochemical purification, recombinant expression, functional assays, CD and NMR studies. Its full-length bioactive form as well as 1–21 and 22–72 fragments (named N(1–21) and C(22–72), respectively) was produced in Escherichia coli by an on-column refolding approach. Recombinant peptide (rMeuTXKβ1) exhibited a low affinity for K⁺ channels and cytolytic effects against bacteria and several eukaryotic cells. N(1–21) was found to preserve anti- Plasmodium activity in contrast to haemolytic activity, whereas C(22–72) retains these two activities. Circular dichroism analysis demonstrates that rMeuTXKβ1 presents a typical scorpion toxin scaffold in water and its α-helical content largely increases in a membrane-mimicking environment, consistent with the NMR structure of N(1–21) and an ab initio structure model of MeuTXKβ1 predicted using I-TASSER algorithm. Our structural and functional data clearly indicate an evolutionary link between TsTXKβ-related peptides and antiparasitic scorpines which both comprise the βSPN (β-KTxs and scorpines) family.

Keywords: Defensin; Antimalarial peptide; Cysteine-stabilized alpha/beta motif; NMR structure; Scorpion venom

Catch muscle of bivalve molluscs contains myosin- and twitchin-associated protein kinase phosphorylating myorod by Oleg S. Matusovsky; Nikolay S. Shelud'ko; Tatyana V. Permyakova; Magdalena Zukowska; Apolinary Sobieszek (pp. 884-890).

We have shown previously that myorod, a molluscan thick filament protein of unknown function, is phosphorylated by vertebrate smooth myosin light chain kinase (MLCK) in N-terminal unique region. The aim of the present study was to clarify whether such phosphorylation may occur in molluscan muscles. We detected three kinases endogenous to molluscan catch muscle, namely, to the complex of surface thick filament proteins that consists of twitchin, myosin, and myorod. The first kinase was a protein kinase A because it was inhibited by a specific inhibitor; the second one was associated with twitchin and phosphorylated myorod at its N-terminal unique region independently of Ca²⁺; and the third kinase was bound to myosin and phosphorylated myorod as well as myosin in the C-terminal part of both proteins. The myosin-associated kinase was inhibited by micromolar concentration of calcium ions. This enzyme could be separated from myosin by chromatography, whereas the kinase associated with twitchin could not be separated from twitchin. Since twitchin has a MLCK-like domain, it is possible that this domain was responsible for myorod phosphorylation. Phosphorylation of myorod within the twitchin–myosin–myorod complex increased the actin-activated Mg²⁺-ATPase activity of myosin. Taken together, these results indicate that phosphorylation of myorod by kinases associated with key proteins of catch contraction may contribute to the functional activity of myorod in molluscan smooth muscle.

Keywords: Abbreviations; MLCK; myosin light chain kinase; PKA; cAMP-dependent protein kinase; TW–MY–MR; a complex of surface proteins from molluscan thick filaments fraction composed of twitchin, myosin and myorod; TW–MR; a complex of twitchin and myorod; MHC; myosin heavy chainsMolluscan catch muscle; Myorod phosphorylation; Myosin; Twitchin; Twitchin kinase

Analysis of proton exchange kinetics with time-dependent exchange rate by Izabela Rutkowska-Wlodarczyk; Borys Kierdaszuk; Jakub Wlodarczyk (pp. 891-898).

Mass spectrometry is used to probe the kinetics of hydrogen–deuterium exchange in lysozyme in pH 5, 6 and 7.4. An analysis based on a Verhulst growth model is proposed and effectively applied to the kinetics of the hydrogen exchange. The data are described by a power-like function which is based on a time-dependence of the exchange rate. Experimental data ranging over many time scales is considered and accurate fits of a power-like function are obtained. Results of fittings show correlation between faster hydrogen–deuterium exchange and increase of pH. Furthermore a model is presented that discriminates between easily exchangeable hydrogens (located in close proximity to the protein surface) and those protected from the exchange (located in the protein interior). A possible interpretation of the model and its biological significance are discussed.

Keywords: Abbreviations; H–D exchange; hydrogen–deuterium exchange; HDX–MS; hydrogen–deuterium exchange coupled with mass spectrometryHydrogen–deuterium exchange; Mass spectrometry; Growth model; Time-dependent exchange rate

Kinetic and biochemical properties of high and low redox potential laccases from fungal and plant origin by Marco Frasconi; Gabriele Favero; Harry Boer; Anu Koivula; Franco Mazzei (pp. 899-908).

The electrochemical studies of laccase–mediator systems are aimed at understanding the mechanism of their redox transformation and their efficiency in both homogeneous and heterogeneous reactions; this topic has paramount application spanning from bleaching of paper pulp and the enzymatic degradation of lignin to the biosensors and biofuel cell development. In this paper four different laccases from Trametes hirsuta (ThL), Trametes versicolor (TvL), Melanocarpus albomyces (r-MaL) and Rhus vernicifera (RvL) were characterized from both biochemical and electrochemical points of view. Two of them (TvL and ThL) are high redox potential and two (RvL and r-MaL) are low redox potential laccases. The outline of this work is focused on the determination of catalytic and bioelectrochemical properties of these four enzymes in homogenous solution as well as immobilized onto electrode surface in the presence of a set of different redox mediators. The results measured in the homogenous reaction system correlated well with those measured with the immobilized enzymes. In addition, they are in good agreement with those reported with reference techniques, suggesting that the electrochemical methods employed in this work can be applied well in place of the traditional techniques commonly used for the kinetic characterization of laccases. These results are also discussed in terms of the known amino acid sequences and three-dimensional (3D) structures of the laccases.

Keywords: Laccase; Mediator; Redox potentials

The glycosylation and characterization of the candidate Gc macrophage activating factor by Tina Ravnsborg; Dorthe T. Olsen; Anna Hammerich Thysen; Maja Christiansen; Gunnar Houen; Hojrup Peter Højrup (pp. 909-917).

The vitamin D binding protein, Gc globulin, has in recent years received some attention for its role as precursor for the extremely potent macrophage activating factor (GcMAF). An O-linked trisaccharide has been allocated to the threonine residue at position 420 in two of the three most common isoforms of Gc globulin (Gc1s and Gc1f). A substitution for a lysine residue at position 420 in Gc2 prevents this isoform from being glycosylated at that position. It has been suggested that Gc globulin subjected sequentially to sialidase and galactosidase treatment generates GcMAF in the form of Gc globulin with only a single GalNAc attached to T420. In this study we confirm the location of a linear trisaccharide on T420. Furthermore, we provide the first structural evidence of the generation of the proposed GcMAF by use of glycosidase treatment and mass spectrometry. Additionally the generated GcMAF candidate was tested for its effect on cytokine release from macrophages in human whole blood.

Keywords: Abbreviations; DHB; 2,5-dihydroxybenzoic acid; FA; Formic acid; Gal; Galactose; GalNAc; N-Acetylgalactosamine; GcMAF; Gc globulin macrophage activating factor; LPS; Lipopolysaccharide; MALDI; Matrix assisted laser desorption ionization; MeCN; Acetonitrile; MeOH; Methanol; PHA; Phytohemagglutinin; Sia; Sialic acid; TFA; Trifluoroacetic acidGc globulin; GcMAF; Glycosylation; Cancer; Glycosidase; Mass spectrometry

Expression proteomics of acute promyelocytic leukaemia cells treated with methotrexate by Nitin Kumar Agarwal; Gerhard Anton Mueller; Claudia Mueller; Jan-Henrick Streich; Abdul Rahman Asif; Hassan Dihazi (pp. 918-928).

Methotrexate was first introduced as a cytotoxic agent that inhibits nucleotide biosynthesis in various cancer disorders; its molecular mechanism remains elusive. To understand the molecular mechanism by which methotrexate induces apoptosis, we analyzed the resulting intracellular protein changes in methotrexate-treated acute promyelocytic leukaemia (HL-60) cells by cysteine-labeled differential in-gel electrophoresis (CL-DIGE) combined with mass spectrometry. Initial CL-DIGE analysis revealed that 24 proteins were differentially expressed ( p<0.05) in the HL-60 cell proteome after treatment with 2.5µM methotrexate for 72h. We found that three structural α4, α5, α7 proteasome subunits, a non-catalytic β3 and two 26S regulatory proteasome subunits were down-regulated in methotrexate-treated HL-60 cells. Western blot analyses further showed that the inhibition of proteasome subunits is accompanied by suppression of NF-κB subunits and promotes the accumulation of ubiquitinated proteins. Furthermore, methotrexate activated unfolded protein response by inducing the expression of endoplasmic reticulum-resident proteins such as calreticulin, protein disulphide isomerase A3 and A4, and 78kDa glucose regulated protein in a time-dependent manner. Altogether, our findings demonstrated that targeting NF-κB, structural and regulatory proteasome subunits with methotrexate may provide new insight into understanding methotrexate-induced apoptotic activities in HL-60 cells.

Keywords: Methotrexate; NF-κB; Proteasome subunits; Cysteine-labeled differential in-gel electrophoresis; Acute promyelocytic leukaemia

Abscisic acid pretreatment enhances salt tolerance of rice seedlings: Proteomic evidence by Xiao-Juan Li; Ming-Feng Yang; Hui Chen; Le-Qing Qu; Fan Chen; Shi-Hua Shen (pp. 929-940).

Enhanced salt tolerance of rice seedlings by abscisic acid (ABA) pretreatment was observed from phenotypic and physiological analyses. Total proteins from rice roots treated with ABA plus subsequent salt stress were analyzed by using proteomics method. Results showed that, 40 protein spots were uniquely upregulated in the seedlings under the condition of ABA pretreatment plus subsequent salt stress, whereas only 16 under the condition of salt treatment. About 78% (31 spots) of the 40 protein spots were only upregulated in the presence of the subsequent salt stress, indicating that plants might have an economical strategy to prevent energy loss under a false alarm. The results also showed that more enzymes involved in energy metabolism, defense, primary metabolism, etc. were upregulated uniquely in ABA-pretreated rice seedlings, suggesting more abundant energy supply, more active anabolism (nitrogen, nucleotide acid, carbohydrate, etc), and more comprehensive defense systems in ABA-pretreated seedlings than in salt stressed ones.

Keywords: Abscisic acid; Proteome; Rice; Salt stress; Tolerance

Interaction ofl-lysine and soluble elastin with the semicarbazide-sensitive amine oxidase in the context of its vascular-adhesion and tissue maturation functions by Aldo Olivieri; Jeff O'Sullivan; Luis Raimon Alvarez Fortuny; Itziar Larrauri Vives; Keith F. Tipton (pp. 941-947).

The copper-containing quinoenzyme semicarbazide-sensitive amine oxidase (EC 1.4.3.21; SSAO) is a multifunctional protein. In some tissues, such as the endothelium, it also acts as vascular-adhesion protein 1 (VAP-1), which is involved in inflammatory responses and in the chemotaxis of leukocytes. Earlier work had suggested that lysine might function as a recognition molecule for SSAO/VAP-1. The present work reports the kinetics of the interaction ofl-lysine and some of its derivatives with SSAO. Binding was shown to be saturable, time-dependent but reversible and to cause uncompetitive inhibition with respect to the amine substrate. It was also specific, sinced-lysine,l-lysine ethyl ester and ε-acetyl-l-lysine, for example, did not bind to the enzyme. The lysine-rich protein soluble elastin bound to the enzyme relatively tightly, which may have relevance to the reported roles of SSAO in maintaining the extracellular matrix (ECM) and in the maturation of elastin. Our data show that lysyl residues are not oxidized by SSAO, but they bind tightly to the enzyme in the presence of hydrogen peroxide. This suggests that binding in vivo of SSAO to lysyl residues in physiological targets might be regulated in the presence of H₂O₂, formed during the oxidation of a physiological SSAO substrate, yet to be identified.

Keywords: Abbreviations; ECM; extracellular matrix; SSAO; semicarbazide-sensitive amine oxidase; VAP; vascular-adhesion proteinSemicarbazide-sensitive amine oxidase (SSAO); Vascular-adhesion protein 1 (VAP 1); l; -Lysine; Elastin; Hydrogen peroxide

Effects of congenital cataract mutation R116H on αA-crystallin structure, function and stability by Min Pang; Jing-Tan Su; Shan Feng; Zhi-Wei Tang; Feng Gu; Meng Zhang; Xu Ma; Yong-Bin Yan (pp. 948-956).

α-crystallin is a molecular chaperone that maintains the optical properties of the lens and delays the onset scattering caused by aging-related protein aggregation. In this research, we found that the missense mutation R116H resulted in an altered size distribution, impaired packing of the secondary structures and modified quaternary structure with great hydrophobic exposure. The mutant exhibited a substrate-dependent chaperone (aggregation–inhibition) or anti-chaperone (aggregation–promotion) effect. Equilibrium unfolding experiments indicated that the mutation stabilized an aggregation-prone intermediate which was not populated during the unfolding of the wild-type protein. The accumulation of this intermediate greatly promoted the formation of non-native large oligomers or aggregates during unfolding. These results suggested that both the aggregation of the mutant upon stress and co-deposition with the target proteins were likely to be responsible for the onset of cataract.

Keywords: αA-crystallin; Autosomal dominant congenital cataract; Missense mutation; Protein stability; Protein aggregationAbbreviations; ANS; 1-anilinonaphtalene-8-sulfonate; CD; circular dichroism; C; _m; GdnHCl concentration at the midpoint of a two-state transition; DTT; dithiothreitol; GdnHCl; guanidine hydrochloride; E; _max; emission maximum wavelength of the intrinsic fluorescence; MW; molecular weight; PAGE; polyacrylamide gel electrophoresis; PBS; phosphate buffered saline; SEC; size-exclusion chromatography; SDS; sodium dodecyl sulfate; sHSP; small heat shock protein; WT; wild-type; [; θ; _MRW; ]; mean residue ellipticity

Biofilm-induced modifications in the proteome of Pseudomonas aeruginosa planktonic cells by Yohan Nigaud; Pascal Cosette; Anthony Collet; Philippe Chan Tchi Song; David Vaudry; Hubert Vaudry; Guy-Alain Junter; Thierry Jouenne (pp. 957-966).

While recent studies focused on Quorum Sensing (QS) role in the cell-to-cell communication in free or biofilm cultures, no work has been devoted up to now to investigate the communication between sessile and planktonic bacteria. In this aim, we elaborated an original two-chambered bioreactor and used a proteomic approach to study the alterations induced by Pseudomonas aeruginosa biofilm cells on protein expression in planktonic counterparts (named SIPs for Surface-Influenced Planktonics). Proteomic analyses revealed the existence of 31 proteins whose amount varied in SIPs, among which five corresponded to hypothetic proteins and two (the Fur and BCP proteins) are involved in bacterial response to oxidative stress. An increase in the concentration of C₄-HSL ( rhlR– rhlI-dependent QS) and 3-oxo-C₁₂-HSL ( lasR– lasI-dependent QS) autoinducer molecules was shown in the planktonic compartment. Interestingly, among proteins that were accumulated by SIPs was 3-oxoacyl-[acyl-carrier-protein] reductase, a protein involved in the production of the autoinducer 3-oxo-C₁₂-HSL. These results demonstrate that planktonic organisms are able to detect the presence of a biofilm in their close environment and to modify their gene expression in consequence.

Keywords: Pseudomonas aeruginosa; Biofilm; Proteome; Surface influenced proteins; Quorum sensing

Biophysical characterization and mutational analysis of the antibiotic resistance protein NimA from Deinococcus radiodurans by Hanna-Kirsti S. Leiros; Bjørn Olav Brandsdal; Seán M. McSweeney (pp. 967-976).

Metronidazole (MTZ) is an antibiotic commonly used to treat anaerobic bacterial infections in humans and animals. Antibiotic resistance toward this class of 5-nitroimidazole (5-Ni) drug derivatives has been related to the Nim genes thought to encode a reductase. Here we report the biophysical characteristics of the NimA protein from Deinococcus radiodurans (DrNimA) binding to MTZ and three other 5-Ni drugs. The interaction energies of the protein and antibiotic are studied by isothermal titration calorimetry (ITC) and with free energy and linear interaction energy (LIE) calculations, where the latter method revealed that the antibiotic binding is mainly of hydrophobic character. ITC measurements further found that one DrNimA dimer has two antibiotic binding sites which were not affected by mutation of the reactive His71. The observed association constants ( K_a) were in the range of 5.1–49⁎10⁴M−¹ and the enthalpy release upon binding to DrNimA for the four drugs studied was relatively low (∼ −1kJ/mol) but still measurable. The drug binding is mainly entropy driven and along with the hydrophobic drug binding site found by crystallography, this possibly explains the low observed enthalpy values. The effect of the His71 mutation and the presence of MTZ were studied by differential scanning calorimetry (DSC). Native DrNimA is a yellow colored protein where the interaction from His71 to the cofactor is thought to be responsible for the coloring. Mutations of His71 to Ala, Ser, Leu or Asp all gave transparent, colorless protein solutions, and the two mutant crystal structures of DrNimA-H71A and DrNimA-H71S presented revealed no cofactor binding.

Keywords: Abbreviations; MD; molecular dynamics; WT; wild type; MS; mass spectroscopy (MS); LIE; linear interaction energy calculations; DrNimA; NimA from; Deinococcus radiodurans; MTZ; metronidazole; TNZ; tinidazole; ONZ; ornidazole; DMZ; dimetridazole; ITC; isothermal titration calorimetry; DSC; differential scanning calorimetryAntibiotic resistance; 5-Nitroimidazole drugs; Isothermal titration calorimetry; Molecular dynamic simulations; Protein structure; Association constants

Mutagenesis and subsite mapping underpin the importance for substrate specificity of the aglycon subsites of glycoside hydrolase family 11 xylanases by Annick Pollet; Stijn Lagaert; Elena Eneyskaya; Anna Kulminskaya; Jan A. Delcour; Christophe M. Courtin (pp. 977-985).

Glycoside hydrolase family (GH) 11 xylanase A from Bacillus subtilis (BsXynA) was subjected to site-directed mutagenesis to probe the role of aglycon active site residues with regard to activity, binding of decorated substrates and hydrolysis product profile. Targets were those amino acids identified to be important by 3D structure analysis of BsXynA in complex with substrate bound in the glycon subsites and the +1 aglycon subsite. Several aromatic residues in the aglycon subsites that make strong substrate–protein interactions and that are indispensable for enzyme activity, were also important for the specificity of the xylanase. In the +2 subsite of BsXynA, Tyr65 and Trp129 were identified as residues that are involved in the binding of decorated substrates. Most interestingly, replacement of Tyr88 by Ala in the +3 subsite created an enzyme able to produce a wider variety of hydrolysis products than wild type BsXynA. The contribution of the +3 subsite to the substrate specificity of BsXynA was established more in detail by mapping the enzyme binding site of the wild type xylanase and mutant Y88A with labelled xylo-oligosaccharides. Also, the length of the cord – a long loop flanking the aglycon subsites of GH11 xylanases – proved to impact the hydrolytic action of BsXynA. The aglycon side of the active site cleft of BsXynA, therefore, offers great potential for engineering and design of xylanases with a desired specificity.

Keywords: Abbreviations; GH; glycoside hydrolase family; BsXynA; Bacillus subtilis; xylanase A; XOS; xylo-oligosaccharides; OSX; oat spelt xylan; X2-9; xylobiose–xylononaose; LVAX; low viscosity wheat flour arabinoxylan; Xylazyme AX; azurine-cross-linked wheat arabinoxylan; XU; xylanase unit; DP; degree of polymerization; AX; arabinoxylans; A/X ratio; arabinose to xylose ratio; HPAEC-PAD; high performance anion exchange chromatography with pulsed amperometric detection; oNP; o-nitrophenolXylanase; Glycoside hydrolase family 11; Substrate specificity; Site-directed mutagenesis; Active site amino acids; Subsite mapping

Critical role of interfaces and agitation on the nucleation of Aβ amyloid fibrils at low concentrations of Aβ monomers by Akiyoshi Morinaga; Kazuhiro Hasegawa; Ryo Nomura; Tadakazu Ookoshi; Daisaku Ozawa; Yuji Goto; Masahito Yamada; Hironobu Naiki (pp. 986-995).

Amyloid deposits are pathological hallmarks of various neurodegenerative diseases including Alzheimer's disease (AD), where amyloid β-peptide (Aβ) polymerizes into amyloid fibrils by a nucleation-dependent polymerization mechanism. The biological membranes or other interfaces as well as the convection of the extracellular fluids in the brain may influence Aβ amyloid fibril formation in vivo. Here, we examined the polymerization kinetics of 2.5, 5, 10 and 20μM Aβ in the presence or absence of air–water interface (AWI) using fluorescence spectroscopy and fluorescence microscopy with the amyloid specific dye, thioflavin T. When the solutions were incubated with AWI and in quiescence, amyloid fibril formation was observed at all Aβ concentrations examined. In contrast, when incubated without AWI, amyloid fibril formation was observed only at higher Aβ concentrations (10 and 20μM). Importantly, when the 5μM Aβ solution was incubated with AWI, a ThT-reactive film was first observed at AWI without any other ThT-reactive aggregates in the bulk. When 5μM Aβ solutions were voltexed or rotated with AWI, amyloid fibril formation was considerably accelerated, where a ThT-reactive film was first observed at AWI before ThT-reactive aggregates were observed throughout the mixture. When 5μM Aβ solutions containing a polypropylene disc were rotated without AWI, amyloid fibril formation was also considerably accelerated, where fine ThT-reactive aggregates were first found attached at the edge of the disc. These results indicate the critical roles of interfaces and agitation for amyloid fibril formation. Furthermore, elimination of AWI may be essential for proper evaluation of the roles of various biological molecules in the amyloid formation studies in vitro.

Keywords: Abbreviations; Aβ; amyloid β-peptide; AWI; air–water interface; AD; Alzheimer's disease; IAPP; islet amyloid peptide; CAA; cerebral amyloid angiopathy; cmc; critical micelle concentration; ThT; thioflavin TAmyloid fibril; Amyloidosis; Amyloid β-peptide; Nucleation; Air–water interface

PONDR-FIT: A meta-predictor of intrinsically disordered amino acids by Bin Xue; Roland L. Dunbrack; Robert W. Williams; A. Keith Dunker; Vladimir N. Uversky (pp. 996-1010).

Protein intrinsic disorder is becoming increasingly recognized in proteomics research. While lacking structure, many regions of disorder have been associated with biological function. There are many different experimental methods for characterizing intrinsically disordered proteins and regions; nevertheless, the prediction of intrinsic disorder from amino acid sequence remains a useful strategy especially for many large-scale proteomic investigations. Here we introduced a consensus artificial neural network (ANN) prediction method, which was developed by combining the outputs of several individual disorder predictors. By eight-fold cross-validation, this meta-predictor, called PONDR-FIT, was found to improve the prediction accuracy over a range of 3 to 20% with an average of 11% compared to the single predictors, depending on the datasets being used. Analysis of the errors shows that the worst accuracy still occurs for short disordered regions with less than ten residues, as well as for the residues close to order/disorder boundaries. Increased understanding of the underlying mechanism by which such meta-predictors give improved predictions will likely promote the further development of protein disorder predictors. Access to PONDR-FIT is available atwww.disprot.org.

Keywords: Abbreviations; IDP; Intrinsically Disordered Protein; IDR; Intrinsically Disordered Region; PDD; Partially Disordered Dataset; PDDS; Structured residues in PDD; PDDM; Residues with Missing electron density in PDD; FOD; Fully Ordered Dataset; FDD; Fully Disordered Dataset; ANN; Artificial Neural Network; ANN-P; ANN trained by PDD; ANN-F; ANN trained by FOD/FDDNatively unfolded; Intrinsically unstructured; Intrinsically disordered; Highly flexible; Highly dynamic; Structurally disordered; Predictor; PONDR

Evolutionary conservation of heavy chain protein transfer between glycosaminoglycans by Kristian W. Sanggaard; Lone Hansen; Carsten Scavenius; Hans-Georg Wisniewski; Torsten Kristensen; Thogersen Ida B. Thøgersen; Jan J. Enghild (pp. 1011-1019).

The bikunin proteins are composed of heavy chains (HCs) covalently linked to a chondroitin sulfate chain originating from Ser-10 of bikunin. Tumor necrosis factor stimulated gene-6 protein (TSG-6)/heavy chain 2 (HC2) cleaves this unique cross-link and transfers the HCs to hyaluronan and other glycosaminoglycans via a covalent HC•TSG-6 intermediate. In the present study, we have investigated if this reaction is evolutionary conserved based on the hypothesis that it is of fundamental importance. The results revealed that plasma/serum samples from mammal, bird, and reptile were able to form TSG-6 complexes suggesting the presence of proteins with the same function as the human bikunin proteins. To substantiate this, the complex forming protein from Gallus gallus (Gg) plasma was purified and identified as a Gg homolog of human HC2•bikunin. In addition, Gg pre-α-inhibitor and smaller amount of high molecular weight forms composed of bikunin and two HCs were purified. Like the human bikunin proteins, the purified Gg proteins were all stabilized by a protein–glycosaminoglycan–protein cross-link, i.e. the HCs were covalently attached to a chondroitin sulfate originating from bikunin. Furthermore, the complex formed between Gg HC2•bikunin and human TSG-6 appeared to be identical to that of the human proteins. Akin to human, Gg HC2 was further transferred to hyaluronan when present, and when incubated in vitro, Gg pre-α-inhibitor and TSG-6, failed to form the intermediate covalent complex, essential for HC transfer. Significantly, Gg HC2, analogous to human HC2, promoted complex formation between human HC3 and human TSG-6, substantiating the evolutionary conservation of these interactions. The present study demonstrates that the unique interactions between bikunin proteins, glycosaminoglycans, and TSG-6 are evolutionary conserved, emphasizing the physiological importance of the TSG-6/HC2-mediated HC-transfer reaction. In addition, the data show that the evolution of HC transfer is likely to predate the role of HC·HA complexes in female fertility and thus has evolved in the context of inflammation rather than fertility.

Keywords: Abbreviations; CS; chondroitin sulfate; GAG; glycosaminoglycan; Gg; Gallus gallus (chicken); HA; hyaluronan; HC; heavy chain; HMW; high molecular weight; IαI; inter-α-inhibitor; MS; mass spectrometry; PαI; pre-α-inhibitor; PGP; protein–glycosaminoglycan–protein; PMF; peptide mass fingerprinting; TSG-6; tumor necrosis factor stimulated gene-6 proteinGlycosaminoglycan; Hyaluronan; Tumor necrosis factor stimulated gene-6 protein (TSG-6); Bikunin proteins; Inter-α-inhibitor (IαI); Extracellular matrix

Development of potent anti-infective agents from Silurana tropicalis: Conformational analysis of the amphipathic, alpha-helical antimicrobial peptide XT-7 and its non-haemolytic analogue [G4K]XT-7 by Anusha P. Subasinghage; J. Michael Conlon; Chandralal M. Hewage (pp. 1020-1028).

Peptide XT-7 (GLLGP⁵LLKIA¹⁰AKVGS¹⁵NLL.NH₂) is a cationic, leucine-rich peptide, first isolated from skin secretions of the frog, Silurana tropicalis (Pipidae). The peptide shows potent, broad-spectrum antimicrobial activity but its therapeutic potential is limited by haemolytic activity (LC₅₀=140µM). The analogue [G4K]XT-7, however, retains potent antimicrobial activity but is non-haemolytic (LC₅₀>500µM). In order to elucidate the molecular basis for this difference in properties, the three dimensional structures of XT-7 and the analogue have been investigated by proton NMR spectroscopy and molecular modelling. In aqueous solution, both peptides lack secondary structure. In a 2,2,2-trifluoroethanol (TFE- d₃)-H₂O mixed solvent system, XT-7 is characterised by a right handed α-helical conformation between residues Leu³ and Leu¹⁷ whereas [G4K]XT-7 adopts a more restricted α-helical conformation between residues Leu⁶ and Leu¹⁷. A similar conformation for XT-7 in 1,2-dihexanoyl- sn-glycero-3-phosphocholine (DHPC) micellular media was observed with a helical segment between Leu³ and Leu¹⁷. However, differences in side chain orientations restricting the hydrophilic residues to a smaller patch resulted in an increased hydrophobic surface relative to the conformation in TFE-H₂O. Molecular modelling of the structures obtained in our study demonstrates the amphipathic character of the helical segments. It is proposed that the marked decrease in haemolytic activity produced by the substitution Gly⁴→Lys in XT-7 arises from a decrease in both helicity and hydrophobicity. These studies may facilitate the development of potent but non-toxic anti-infective agents based upon the structure of XT-7.

Keywords: Abbreviations; CD; circular dichroism; DHPC; 1,2-dihexanoyl-; sn; -glycero-3-phosphocholine; MIC; minimum inhibitory concentration; NMR; nuclear magnetic resonance; NOE; nuclear Overhauser effect; NOESY; nuclear Overhauser effect spectroscopy; TFE; trifluoroethanol; TOCSY; total correlation spectroscopy; RMSD; root mean square deviation Silurana tropicalis; Antimicrobial; XT-7; Amphipathic α-helix; NMR

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BBA - Proteins and Proteomics (v.1804, #4)