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BBA - Proteins and Proteomics (v.1784, #5)

Editorial Board (pp. ii).

Functional mechanics of the ATP-dependent Lon protease- lessons from endogenous protein and synthetic peptide substrates by Irene Lee; Carolyn K. Suzuki (pp. 727-735).

Lon, also known as the protease La, is a homo-oligomeric ATP-dependent protease, which is highly conserved in archaea, eubacteria and eukaryotic mitochondria and peroxisomes. Since its discovery, studies have shown that Lon activity is essential for cellular homeostasis, mediating protein quality control and metabolic regulation. This article highlights the discoveries made over the past decade demonstrating that Lon selectively degrades abnormal as well as certain regulatory proteins and thus plays significant roles in maintaining bacterial and mitochondrial function and integrity. In addition, Lon is required in certain pathogenic bacteria, for rendering pathogenicity and host infectivity. Recent research endeavors have been directed toward elucidating the reaction mechanism of the Lon protease by different biochemical and structural biological techniques. In this mini-review, the authors survey the diverse biological roles of Lon, and also place special emphasis on recent findings that clarify the mechanistic aspects of the Lon reaction cycle.

Keywords: Abbreviations; ATP; adenosine triphosphate; ADP; adenosine diphosphate; AMPPNP; adenylyl 5-imidodiphosphate; Abz; anthranilamide; Bz; benzoic acid; MANT; 2'-(or 3'); O; -(; N; -methylanthraniloyl); Dansyl; 5-dimethylamino-1-naphthalenesulfonyl; FITC; fluorescein isothiocyanate; Abu; amninobutyric acidOligomeric ATP-dependent proteases; Protein; Peptide; Mechanism

Thermodynamic and kinetic stability of penicillin acylase from Escherichia coli by Valerij Ya. Grinberg; Tatiana V. Burova; Natalia V. Grinberg; Tatiana A. Shcherbakova; Dorel T. Guranda; Ghermes G. Chilov; Vytas K. Švedas (pp. 736-746).

Thermal denaturation of penicillin acylase (PA) from Escherichia coli has been studied by high-sensitivity differential scanning calorimetry as a function of heating rate, pH and urea concentration. It is shown to be irreversible and kinetically controlled. Upon decrease in the heating rate from 2 to 0.1 K min−¹ the denaturation temperature of PA at pH 6.0 decreases by about 6 °C, while the denaturation enthalpy does not change notably giving an average value of 31.6±2.1 J g−¹. The denaturation temperature of PA reaches a maximum value of 64.5 °C at pH 6.0 and decreases by about of 15 °C at pH 3.0 and 9.5. The pH induced changes in the denaturation enthalpy follow changes in the denaturation temperature. Increasing the urea concentration causes a decrease in both denaturation temperature and enthalpy of PA, where denaturation temperature obeys a linear relation. The heat capacity increment of PA is not sensitive to the heating rate, nor to pH, and neither to urea. Its average value is of 0.58±0.02 J g−¹ K−¹. The denaturation transition of PA is approximated by the Lumry–Eyring model. The first stage of the process is assumed to be a reversible unfolding of the α-subunit. It activates the second stage involving dissociation of two subunits and subsequent denaturation of the β-subunit. This stage is irreversible and kinetically controlled. Using this model the temperature, enthalpy and free energy of unfolding of the α-subunit, and a rate constant of the irreversible stage are determined as a function of pH and urea concentration. Structural features of the folded and unfolded conformation of the α-subunit as well as of the transition state of the PA denaturation in aqueous and urea solutions are discussed.

Keywords: Penicillin acylase; Denaturation; Thermodynamics; Kinetics; HS-DSC

MH1 domain of SMAD4 binds N-terminal residues of the homeodomain of Hoxc9 by Bo Zhou; Lihong Chen; Xing Wu; Jing Wang; Yinliang Yin; Guang Zhu ⁎ (pp. 747-752).

Smad family proteins mediate signaling initiated by bone morphogenetic proteins (BMPs). Upon BMP stimulation, the Smads such as Smad4 can interact directly with Hox proteins and suppress their DNA-binding activity. Although the interaction between the MAD-homology 1 (MH1) domain of Smad4 and Hox was found to regulate the transcription activity of Hox proteins, the molecular mechanism is not well characterized and direct contact residues remain to be elucidated. In the present study, the interaction between the recombinant homeodomain (HD) of Hoxc9 and MH1 domain of Smad4 was investigated with the use of the GST pull-down assay, surface plasmon resonance (SPR) analysis as well as multidimensional nuclear magnetic resonance (NMR) techniques. The Hoxc9-HD was precipitated with the GST-fused Smad4-MH1 but not with GST alone, demonstrating a direct interaction between Hoxc9-HD and Smad4-MH1 in vitro. SPR measurement further confirmed a moderately strong interaction ( K_d≈400 nM) between these two domains. Moreover, NMR titration experiments showed that a strong and specific binding occurred between Smad4-MH1 and Hoxc9-HD. NMR triple-resonance experiments and backbone assignments revealed that the N-terminal arm of Hoxc9-HD, spanning the positive-charged DNA-binding segment of Arg190–Arg196, was intimately involved in the interaction with Smad4-MH1. Ala-substitutions of Arg190–Arg196 led to the loss of interaction between Hoxc9-HD and Smad4-MH1 in both GST-pull down assay and SPR analysis; further provided functional evidence for the critical role of this positive-charged region in binding to Smad4-MH1. This suggested that Smad4-MH1 could occupy one of the DNA binding sites of Hoxc9 and consequently inhibits its transcription activity. The above results are in good agreement and yield the first insight into the interaction between the homeodomain of Hox proteins and the conserved MH1 domain of Smad family proteins.

Keywords: MH1; SMAD4; Homeodomain; Hoxc9; Protein–protein interaction; NMR

Identification of a plasmin-interactive site within the A2 domain of the factor VIII heavy chain by Keiji Nogami; Katsumi Nishiya; Evgueni L. Saenko; Masahiro Takeyama; Ichiro Tanaka; Akira Yoshioka; Midori Shima (pp. 753-763).

Factor VIII is activated and inactivated by plasmin by limited proteolysis. In our one-stage clotting assay, these plasmin-catalyzed reactions were inhibited by the addition of isolated factor VIII A2 subunits and by Glu-Gly-Arg-active-site modified factor IXa. SDS-PAGE analysis showed that an anti-A2 monoclonal antibody, recognizing the factor IXa-interactive site (residues 484–509), blocked the plasmin-catalyzed cleavage at Arg³³⁶ and Arg³⁷² but not at Arg⁷⁴⁰. Surface plasmon resonance-based assays and ELISA demonstrated that the A2 subunit bound to active-site modified anhydro-plasmin with high affinity ( K_d: 21 nM). Both an anti-A2 monoclonal antibody and a peptide comprising of A2 residues 479–504 blocked A2 binding by ∼80% and ∼55%, respectively. Mutant A2 molecules where the basic residues in A2 were converted to alanine were evaluated for binding of anhydro-plasmin. Among the tested mutants, the R484A A2 mutant possessed ∼250-fold lower affinity than the wild-type A2. The affinities of K377A, K466A, and R471A mutants were decreased by 10–20-fold. The inhibitory effect of R484A mutant on plasmin-catalyzed inactivation of factor VIIIa was ∼20% of that of wild-type A2. In addition, the inactivation rate by plasmin of factor VIIIa reconstituted with R484A mutant was ∼3-fold lower than that with wild-type A2. These findings demonstrate that Arg⁴⁸⁴ plays a key role within the A2 plasmin-binding site, responsible for plasmin-catalyzed factor VIII(a) inactivation.

Keywords: Abbreviations; APC; activated protein C; mAb; monoclonal antibody; EGR-factor IXa; Glu-Gly-Arg-active-site modified factor IXa; 6-AHA; 6-aminohexanoic acid; Ah-plasmin; anhydro-plasmin; wild-type A2; wt-A2; SPR-based assay; surface plasmon resonance-based assay; LRP; low-density lipoprotein receptor-related proteinFactor VIII; A2 domain; Plasmin; Interactive-site; Inactivation; A2 mutant

Protein expression profile characteristic to hepatocellular carcinoma revealed by 2D-DIGE with supervised learning by Reiji Teramoto; Hirotaka Minagawa; Masao Honda; Kenji Miyazaki; Yo Tabuse; Ken'ichi Kamijo; Teruyuki Ueda; Shuichi Kaneko (pp. 764-772).

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies. Although several major risks related to HCC, e.g., hepatitis B and/or hepatitis C virus infection, aflatoxin B1 exposure, alcohol drinking and genetic defects have been revealed, the molecular mechanisms leading to the initiation and progression of HCC have not been clarified. To reduce the mortality and improve the effectiveness of therapy, it is important to detect the proteins which are associated with tumor progression and may be useful as potential therapeutic or diagnosis targets. However, previous studies have not yet revealed the associations among HCC cells, histological grade and AFP. Here, we performed two-dimensional difference gel electrophoresis (2D-DIGE) combined with MS for 18 HCC patients. To focus not on individual proteins but on multiple proteins associated with pathogenesis, we introduce the supervised feature selection based on stochastic gradient boosting (SGB) for identifying protein spots that discriminate HCC/non HCC, histological grade of moderate/well and high α-fetoprotein (AFP)/low AFP level without arbitrariness. We detected 18, 25 and 27 protein spots associated with HCC, histological grade and AFP level, respectively. We confirmed that SGB is able to identify the known HCC-related proteins, e.g., heat shock proteins, carbonic anhydrase 2. Moreover, we identified the differentially expressed proteins associated with histological grade of HCC and AFP level and found that aldo-keto reductase 1B10 (AKR1B10) is related to well differentiated HCC, keratin 8 (KRT8) is related to both histological grade and AFP level and protein disulfide isomerase-associated 3 (PDIA3) is associated with both HCC and AFP level. Our pilot study provides new insights on understanding the pathogenesis of HCC, histological grade and AFP level.

Keywords: Hepatocelluar carcinoma; Two-dimensional difference gel electrophoresis; Supervised learning; Protein profiling; Feature selection

Proteomic identification of differentially expressed genes in mouse neural stem cells and neurons differentiated from embryonic stem cells in vitro by Kuniko Akama; Ryosuke Tatsuno; Masahiro Otsu; Tomoe Horikoshi; Takashi Nakayama; Megumi Nakamura; Tosifusa Toda; Nobuo Inoue (pp. 773-782).

Embryonic stem (ES) cells are pluripotent stem cells and give rise to a variety of differentiated cell types including neurons. To study a molecular basis for differentiation from ES cells to neural cells, we searched for proteins involved in mouse neurogenesis from ES cells to neural stem (NS) cells and neurons by two-dimensional gel electrophoresis (2-DE) and peptide mass fingerprinting, using highly homogeneous cells differentiated from ES cells in vitro. We newly identified seven proteins with increased expression and one protein with decreased expression from ES cells to NS cells, and eight proteins with decreased expression from NS cells to neurons. Western blot analysis confirmed that a tumor-specific transplantation antigen, HS90B, decreased, and an extracellular matrix and membrane glycoprotein (such as laminin)-binding protein, galectin 1 (LEG1), increased in NS cells, and LEG1 and a cell adhesion receptor, laminin receptor (RSSA), decreased in neurons. The results of RT-PCR showed that mRNA of LEG1 was also up-regulated in NS cells and down-regulated in neurons, implying an important role of LEG1 in regulating the differentiation. The differentially expressed proteins identified here provide insight into the molecular basis of neurogenesis from ES cells to NS cells and neurons.

Keywords: Embryonic stem cell; Differential expression; Neural stem cell; Neuronal differentiation

Structural and functional characterization of osmotically inducible protein C (OsmC) from Thermococcus kodakaraensis KOD1 by Seong-Cheol Park; Bang Phuong Pham; Le Van Duyet; Baolei Jia; Sangmin Lee; Rui Yu; Sang Woo Han; Jae-Kyung Yang; Kyung-Soo Hahm; Gang-Won Cheong (pp. 783-788).

Osmotically inducible protein C (OsmC) is involved in the cellular defense mechanism against oxidative stress caused by exposure to hyperoxides or elevated osmolarity. OsmC was identified by two-dimensional electrophoresis (2DE) analysis as a protein that is overexpressed in response to osmotic stress, but not under heat and oxidative stress. Here, an OsmC gene from T. kodakaraensis KOD1 was cloned and expressed in Escherichia coli. TkOsmC showed a homotetrameric structure based on gel filtration and electron microscopic analyses. TkOsmC has a significant peroxidase activity toward both organic and inorganic peroxides in high, but not in low temperature.

Keywords: Archaeon; Thermococcus kodakaraensis; KOD1; OsmC; Electron microscopy

Flexibility and enzyme activity of NADH oxidase from Thermus thermophilus in the presence of monovalent cations of Hofmeister series by Kamil Tóth; Erik Sedlák; Mathias Sprinzl; Gabriel Žoldák (pp. 789-795).

Recently, we have shown that anions of Hofmeister series affect the enzyme activity through modulation of flexibility of its active site. The enzyme activity vs. anion position in Hofmeister series showed an unusual bell-shaped dependence. In the present work, six monovalent cations (Na⁺, Gdm⁺, NH₄⁺, Li⁺, K⁺ and Cs⁺) of Hofmeister series with chloride as a counterion have been studied in relation to activity and stability of flavoprotein NADH oxidase from Thermus thermophilus (NOX). With the exception of strongly chaotropic guanidinium cation, cations are significantly less effective in promoting the Hofmeister effect than anions mainly due to repulsive interactions of positive charges around the active site. Thermal denaturations of NOX reveal unfavorable electrostatic interaction at the protein surface that may be shielded to different extent by salts. Michaelis–Menten constants for NADH, accessibility of the active site as reflected by Stern–Volmer constants and activity of NOX at high cation concentrations (1–2 M) show bell-shaped dependences on cation position in Hofmeister series. Our analysis indicates that in the presence of kosmotropic cations the enzyme is more stable and possibly more rigid than in the presence of chaotropic cations. Molecular dynamic (MD) simulations of NOX showed that active site switches between open and closed conformations [J. Hritz, G. Zoldak, E. Sedlak, Cofactor assisted gating mechanism in the active site of NADH oxidase from Thermus thermophilus, Proteins 64 (2006) 465–476]. Enzyme activity, as well as substrate binding, can be regulated by the salt mediated perturbation of the balance between open and closed forms. We propose that compensating effect of accessibility and flexibility of the enzyme active site leads to bell-shaped dependence of the investigated parameters.

Keywords: Abbreviations; NOX; NADH oxidase; MD; molecular dynamics; K; _M,NADH; and; K; _M,FMN; Michaelis–Menten constant for NADH and FMN, respectively; K; _SV; Stern–Volmer constant,; T; _trs; transition temperatureHofmeister effect; NADH oxidase; Gating mechanism; Active site; Fluorescence quenching

Conformational behavior of polypeptides derived through simultaneous global conservative site-directed mutagenesis of chymotrypsin inhibitor 2 by Shubbir Ahmed; Divya Kapoor; Balvinder Singh; Purnananda Guptasarma (pp. 796-805).

The natural occurrence of conservative residue substitutions in proteins suggests that side-chain packing schemes in protein interiors can accommodate mutational replacements of residues by others of similar nature. To explore the extent to which such substitutions are tolerated, especially when introduced simultaneously and globally over the entire length of a polypeptide chain, we examined the conformational behavior of a model 65 residues-long protein, wild-type chymotrypsin inhibitor 2 (WTCI2), and two globally-mutated (GM) variants named GMCI2-1 and GMCI2-2, each incorporating 55 conservative residue substitutions. GMCI2-1, was soluble over a wide range of pH, and folded into a compact, spherical, monomer marked by (i) complete absence of surface hydrophobicity, (ii) a WTCI2-like betaII-type CD spectrum, (iii) high WTCI2-like thermal stability, and (d) 1D and 2D NMR spectra characteristic of folded protein structure. GMCI2-2 was insoluble over a wide range of pH, and could be solubilized only at pH 4.0, showing non-WTCI2-like far-UV CD spectra characterized by high helical content. These results tentatively indicate that polypeptides incorporating residues of identical nature at equivalent chain locations can show the potential to fold with similar characteristics. However, further detailed investigations would be required to determine whether indeed the structural fold of GMCI2-1 resembles that of WTCI2, and to evaluate the extent to which it does so.

Keywords: Protein folding; Inverse protein folding; Protein engineering; Protein sequence–structure relationships

Thermodynamic analysis ofl-arginine and N^ω-hydroxy-l-arginine binding to nitric oxide synthase by Henrik Zakariassen; F. Henning Cederkvist; Espen Harbitz; Toru Shimizu; Reinhard Lange; Bernd Mayer; Antonius C.F. Gorren; K. Kristoffer Andersson; Sorlie Morten Sørlie (pp. 806-810).

Isothermal titration calorimetry has been used to determine thermodynamic parameters of substrate binding to the oxygenase domain of neuronal nitric oxide synthase (nNOS_oxy) in the presence of the cofactor tetrahydrobiopterin. The intermediate N^ω-hydroxy-l-arginine (NHA) has a larger affinity thanl-Arginine (l-Arg) for nNOS_oxy, with K_d=0.4±0.1 µM and 1.7±0.3 µM at 25 °C, respectively. nNOS_oxy binds NHA andl-Arg with Δ H −4.1±0.2 and −1.0±0.1 kcal/mol and Δ S=15 and 23 cal/Kmol respectively. NHA binding is more exothermic probably due to formation of an extra hydrogen bond in the active site compared tol-Arg. The changes in heat capacity (Δ C_p) are relatively small for binding of both NHA andl-Arg (−53±18 and −95±23 cal/L mol, respectively), which indicates that hydrophobic interactions contribute little to binding.

Keywords: Nitric oxide synthase; ITC; Thermodynamics; Binding

Converting human carbonic anhydrase II into a benzoate ester hydrolase through rational redesign by Gunnar E. Höst; Bengt-Harald Jonsson (pp. 811-815).

Enzymes capable of benzoate ester hydrolysis have several potential medical and industrial applications. A variant of human carbonic anhydrase II (HCAII) was constructed, by rational design, that is capable of hydrolysing para-nitrophenyl benzoate (pNPBenzo) with an efficiency comparable to some naturally occurring esterases. The design was based on a previously developed strategy [G. Höst, L.G. Mårtensson, B.H. Jonsson, Redesign of human carbonic anhydrase II for increased esterase activity and specificity towards esters with long acyl chains, Biochim. Biophys. Acta 1764 (2006) 1601–1606.], in which docking of a transition state analogue (TSA) to the active site of HCAII was used to predict mutations that would allow the reaction. A triple mutant, V121A/V143A/T200A, was thus constructed and shown to hydrolyze pNPBenzo with k_cat/ K_M=625 (± 38) M⁻¹ s⁻¹. It is highly active with other ester substrates as well, and hydrolyzes para-nitrophenyl acetate with k_cat/ K_M=101,700 (± 4800) M⁻¹ s⁻¹, which is the highest esterase efficiency so far for any CA variant. A parent mutant (V121A/V143A) has measurable K_M values for para-nitrophenyl butyrate (pNPB) and valerate (pNPV), but for V121A/V143A/T200A no K_M could be determined, showing that the additional T200A mutation has caused a decreased substrate binding. However, k_cat/ K_M is higher with both substrates for the triple mutant, indicating that binding energy has been diverted from substrate binding to transition state stabilization.

Keywords: Carbonic anhydrase; Hydrolysis; Specificity; Mutagenesis; Protein engineering; Rational design

Glutathionylation of the iron superoxide dismutase from the psychrophilic eubacterium Pseudoalteromonas haloplanktis by Immacolata Castellano; Maria Rosaria Ruocco; Francesca Cecere; Antimo Di Maro; Angela Chambery; Andzelika Michniewicz; Giuseppe Parlato; Mariorosario Masullo; Emmanuele De Vendittis (pp. 816-826).

Our previous work showed that the adduct between β-mercaptoethanol and the single cysteine residue (Cys57) in superoxide dismutase from the psychrophilic eubacterium Pseudoalteromonas haloplanktis ( PhSOD) reduces the enzyme inactivation by peroxynitrite. In this work, immunoblotting experiments prove that peroxynitrite inactivation of PhSOD involves formation of nitrotyrosine residue(s). In order to study the role of Cys57 as a redox-sensor residue modifiable by cellular thiols, a recombinant PhSOD and two Cys57 mutants were produced and characterized. Recombinant and mutant enzymes share similar activity and peroxynitrite inactivation, but different reactivity towards three glutathione forms. Indeed, oxidized glutathione and S-nitrosoglutathione, but reduced glutathione, lead to S-glutathionylation of recombinant PhSOD. This new covalent modification for a Fe-SOD does not occur in both Cys57 mutants, thus indicating that its target is Cys57. Moreover, mass spectrometry analysis confirmed that S-glutathionylation of Cys57 takes place also with endogenous PhSOD. Formation of this mixed disulfide in PhSOD protects the enzyme from tyrosine nitration and peroxynitrite inactivation. PhSOD undergoes S-glutathionylation during its overproduction in E. coli cells and in a growing culture of P. haloplanktis. In both cases the extent of glutathionylated PhSOD is enhanced upon cell exposure to oxidative agents. We suggest that S-glutathionylation of PhSOD could represent a further cold-adaptation strategy to improve the antioxidant cellular defence mechanism.

Keywords: Abbreviations; GSH; reduced glutathione; GSNO; S-nitrosoglutathione; GSSG; oxidized glutathione; IC; ₅₀; inactivator concentration causing 50% reduction of activity; IPTG; isopropyl-β-thiogalactopyranoside; PBS; phosphate buffer saline; Ph; SOD; SOD from; Pseudoalteromonas haloplanktis; Ph; SOD; _rec; recombinant; Ph; SOD; PVDF; poly (vinylidene difluoride); ROS; reactive oxygen species; SOD; superoxide dismutaseS-glutathionylation; Superoxide dismutase; Pseudoalteromonas haloplanktis; Psychrophile; Peroxynitrite

Locating the rate-determining step(s) for three-step hydrolase-catalyzed reactions with dynafit by Daoning Zhang; Ildiko M. Kovach; John Paul Sheehy (pp. 827-833).

Hydrolytic reactions of oligopeptide 4-nitroanilides catalyzed by human-α-thrombin, human activated protein C and human factor Xa were studied at pH 8.0–8.4 and 25.0±0.1 °C by the progress curve method and individual rate constants were calculated mostly within 10% internal error using DYNAFITV. A systematic strategy has been developed for fitting a three-step consecutive mechanism to eighteen hundred to six thousand time-course data points polled from two to four independent kinetic experiments. Enzyme and substrate concentrations were also calculated. Individual rate constants well reproduce published values obtained under comparable conditions and the Michaelis–Menten kinetic parameters calculated from these elementary rate constants are also within reasonable limits of published values. For comparison, the integrated Michaelis–Menten equation was also fitted to data from twelve sets. Both the k_cat and k_cat/ K_m values are within 15% agreement with those calculated using the elementary rate constants obtained with DYNAFITV. Rate constants for the second and third consecutive steps are within 3–4 fold indicating that both determine the overall rate. The Factor Xa-catalyzed hydrolysis of N-α-Z-d-Arg-Gly-Arg-pNA·2HCl at pH 8.4 in a series of buffers containing increasing fractions of deuterium at 25.0±0.1 °C shows a very strong dependence of k₃ and a moderate dependence of k₂ on D content in the buffer: the fractionation factors are: 0.49±0.03 for K_1, 0.70±0.05 for k₂, and (0.32±0.03)² for k_3.

Keywords: Enzyme kinetics; Progress curve; Elementary rate constants; Blood cascade enzymes; Solvent isotope effects; Proton inventories

Dynamics of oligomer formation by denatured carbonic anhydrase II by Dmitry A. Prokhorov; Alexander A. Timchenko; Vladimir N. Uversky; Vladimir S. Khristoforov; Hiroshi Kihara; Kazumoto Kimura; Viktor P. Kutyshenko (pp. 834-842).

Aggregation and subsequent development of protein deposition diseases originate from conformational changes in corresponding amyloidogenic proteins. Many proteins unrelated to amyloidoses also fibrillate at the appropriate conditions. These proteins serve as a model for studying the processes of protein misfolding, oligomerization and fibril formation. The accumulated data support the model where protein fibrillogenesis proceeds via the formation of a relatively unfolded amyloidogenic conformation. The urea-induced unfolding of bovine carbonic anhydrase II, BCA II, is characterized by a combination of high-resolution NMR, circular dichroism spectroscopy and small angle X-ray scattering. It is shown that the formation of associates of protein molecules in complex with solvent (water and urea), APS, takes place in the presence of 4–6 M urea. The subsequent increase in urea concentration to 8 M is accompanied by a disruption of APS and leads to a complete unfolding of a protein molecule. Analysis of BCA II self-association in the presence of 4.2 M urea revealed that APS are relatively large mostly β-structural blocks with the averaged molecular mass of 190–220 kDa. This work also demonstrates some novel NMR-based methodological approaches that provide useful information on protein self-association.

Keywords: Abbreviations; SD; spin diffusion; SSD; spectrum of spin diffusion; 1M-spectrum; reference; ¹; H NMR spectrum; APS; associates of protein and solvent molecules; offRI; off-resonance irradiation; NMR; nuclear magnetic resonance; CD; circular dichroism; UV; ultra violet; SAXS; small angle X-ray scattering; BCA II; bovine carbonic anhydrase IICarbonic anhydrase II; NMR; Circular dichroism; Small angle X-ray scattering; Spin diffusion; Protein association; Amyloid-like structure

The role of the sulfonium linkage in the stabilization of the ferrous form of myeloperoxidase: A comparison with lactoperoxidase by Silvia Brogioni; Johanna Stampler; Furtmuller Paul G. Furtmüller; Alessandro Feis; Christian Obinger; Giulietta Smulevich (pp. 843-849).

In all mammalian peroxidases, the heme is covalently attached to the protein via two ester linkages between conserved aspartate (Asp94) and glutamate residues (Glu242) and modified methyl groups on pyrrole rings A and C. Only myeloperoxidase has an additional sulfonium ion linkage between the sulfur atom of the conserved methionine 243 and the β-carbon of the vinyl group on pyrrole ring A. Upon reduction from Fe(III) to Fe(II), lactoperoxidase (LPO) but not myeloperoxidase (MPO) is shown to adopt three distinct active site conformations which depend on pH and time. Comparative spectroscopic analysis (UV-Vis absorption and resonance Raman) of the ferrous forms of LPO, wild-type MPO and the variants Asp94Val, Glu242Gln, Met243Thr and Met243Val clearly demonstrate that a single, stable ferrous form of MPO is present only in those proteins which retain an intact sulfonium linkage. By contrast, both ferrous Met243Thr and Met243Val can assume two conformations. They resemble ferrous LPO, being five-coordinated high-spin species that are distinguished by the strength of the proximal Fe-histidine bond. This bond weakens with time or decreasing pH, as indicated by the Fe-histidine stretching bands.

Keywords: Abbreviations; MPO; myeloperoxidase; LPO; lactoperoxidase; WT; wild type; recMPO; recombinant MPO; DMSO; dimethyl sulfoxide; RR; resonance Raman; D2; second derivative; 5c; five- coordinate; 6c; six-coordinate; HS; high-spin; LS; low-spin; CD; circular dichroism; E; '°; standard reduction potentialMyeloperoxidase; Lactoperoxidase; Ferrous form; Heme to protein linkage; Resonance Raman spectroscopy; Fe-Im stretching frequency; Electronic absorption; Site-directed mutagenesis

A Kunitz trypsin inhibitor of Entada scandens seeds: Another member with single disulfide bridge by M.H. Lingaraju; Lalitha R. Gowda (pp. 850-855).

Sword bean ( Entada scandens) is a tree climber that belongs to Mimosoideae, a subfamily of Leguminosae. A potent Kunitz type trypsin inhibitor (ESTI) was purified to homogeneity from Entada scandens seeds by sequential ammonium sulfate precipitation, affinity chromatography on trypsin-Sepharose and DEAE-Sephacel ion-exchange chromatography. ESTI is a single polypeptide chain of 19,766 Da. Both native PAGE as well as isoelectric focusing showed a single inhibitor species with a pI of 7.43. MALDI-TOF analysis also confirmed the monomeric nature. The amino-terminal sequence of ESTI reveals significant homology to the Kunitz-type protease inhibitors of legume plants. ESTI is unique in that it contains a single disulfide bridge, and unlike other inhibitors from Mimosoideae species is a single chain polypeptide. ESTI inhibited bovine trypsin with a stoichiometry of 1:1 and the apparent K_i was 4.9×10−⁹ M. In vitro assay showed that ESTI inhibited the midgut proteinase of the fifth instar larvae of Rice moth ( Corcyra cephalonica) with an IC₅₀ of 26.4±0.01 nM. ESTI exhibits a mixed type competitive inhibition at lower concentration and pure competitive at higher inhibitor concentrations. Phylogenetic analyses depicted a clear divergence of single disulfide containing inhibitors from other tree legume Kunitz inhibitors. The homology of ESTI to Kunitz inhibitors together with the absence of Bowman-Birk type inhibitors in sword bean further supports the theory that there exists an evolutionary relationship between the families of inhibitors found in Leguminosae.

Keywords: Abbreviations; APNE; N; -acetyl-; dl; -phenylalanine-β-naphthyl ester; BAPNA; N; -α-benzoyl-; dl; -arginine-; p; -nitroanilide; BTPNA; N; -benzoyl-; l; -tyrosine-; p; -nitroanilide; BBI; Bowman-Birk inhibitor; CMGP; Corcyra cephalonica; midgut proteinase; DMSO; Dimethyl sulfoxide; ESTI; Entada scandens; trypsin inhibitor; KTI; Kunitz trypsin inhibitor; MALDI-TOF-MS; Matrix Assisted Laser Desorption Ionization–Time of Flight–Mass Spectroscopy; TPCK; l; -1-tosylamido-2-phenylalanine chloromethyl ketone; TIU; Trypsin inhibitory unitMimosoideae; Sword bean; Proteinase inhibitor; Mixed type competitive inhibition; Multiple alignment; Evolutionary theory

Point mutations abolishing the mannose-binding capability of boar spermadhesin AQN-1 by Mahnaz Ekhlasi-Hundrieser; Juan J. Calvete; Bettina Von Rad; Christiane Hettel; Manfred Nimtz; Topfer-Petersen Edda Töpfer-Petersen (pp. 856-862).

The mannose-binding capability of recombinant wild-type boar spermadhesin AQN-1 and of its site-directed mutants in the highly-conserved region around of the single glycosylation site (asparagine 50) of some spermadhesins, where the carbohydrate binding site has been proposed to be located, was checked using a solid-phase assay and a biotinylated mannose ligand. Substitution of glycine 54 by amino acids bearing an unipolar side chain did not cause significant decrease in the mannose-binding activity. However, amino acids with uncharged polar side chains or having a charged polar side chain abolished the binding of biotinylated mannose to the corresponding AQN-1 mutants. The results suggest that the higher surface accessibility of amino acids possessing polar side chains compared to those bearing nonpolar groups may sterically interfere with monosaccharide binding. The location of the mannose-binding site in AQN-1 appears to be topologically conserved in other heparin-binding boar spermadhesins, i.e., AQN-3 and AWN, but departs from the location of the mannose-6-phosphate-recognition site of PSP-II. This indicates that different spermadhesin molecules have evolved non-equivalent carbohydrate-binding capabilities, which may underlie their distinct patterns of biological activities.

Keywords: Seminal plasma protein; Spermadhesin; AQN-1; Acidic seminal fluid; Protein; Carbohydrate-binding protein; CUB domain

Role of tryptophan-208 residue in cytochrome c oxidation by ascorbate peroxidase from Leishmania major-kinetic studies on Trp208Phe mutant and wild type enzyme by Rajesh K. Yadav; Subhankar Dolai; Swati Pal; Subrata Adak (pp. 863-871).

Ascorbate peroxidase from L. Major (LmAPX) is a functional hybrid between cytochrome c peroxidase (CCP) and ascorbate peroxidase (APX). We utilized point mutagenesis to investigate if a conserved proximal tryptophan residue (Trp208) among Class I peroxidase helps in controlling catalysis. The mutant W208F enzyme had no effect on both apparent dissociation constant of the enzyme–cytochrome c complex and K_m value for cytochrome c indicating that cytochrome c binding affinity to the enzyme did not alter after mutation. Surprisingly, the mutant was 1000 times less active than the wild type in cytochrome c oxidation without affecting the second order rate constant of compound I formation. Our diode array stopped-flow spectral studies showed that the substrate unbound wild type enzyme reacts with H₂O₂ to form compound I (compound II type spectrum), which was quite different from that of compound I in W208F mutant as well as horseradish peroxidase (HRP). The spectrum of the compound I in wild type LmAPX showed a red shift from 409 nm to 420 nm with equal intensity, which was broadly similar to those of known Trp radical. In case of compound I for W208F mutant, the peak in the Soret region was decreased in heme intensity at 409 nm and was not shifted to 420 nm suggesting this type of spectrum was similar to that of the known porphyrin π-cation radical. In case of an enzyme–H₂O₂–ascorbate system, the kinetic for formation and decay of compound I and II of a mutant enzyme was almost identical to that of a wild type enzyme. Thus, the results of cytochrome c binding, compound I formation rate and activity assay suggested that Trp208 in LmAPX was essential for electron transfer from cytochrome c to heme ferryl but was not indispensable for ascorbate or guaiacol oxidation.

Keywords: Abbreviations; APX; ascorbate peroxidase; LmAPX; Leishmania major; ascorbate peroxidase; HRP; horseradish peroxidase; CCP; cytochrome; c; peroxidaseLeishmania; Peroxidase; Cytochrome; c; Electron transfer; Rapid kinetics

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BBA - Proteins and Proteomics (v.1784, #5)

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BBA - Proteins and Proteomics (v.1784, #5)