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BBA - Proteins and Proteomics (v.1774, #7)

Editorial Board (pp. ii).

Helicity propensity and interaction of synthetic peptides from heptad-repeat domains of herpes simplex virus 1 glycoprotein H: A circular dichroism study by Barbara Sanavio; Angela Piccoli; Tatiana Gianni; Carlo Bertucci (pp. 781-791).

The secondary structure of two synthetic peptides from heptad-repeat domains of herpes simplex virus 1 glycoprotein H was determined by circular dichroism. In particular, the propensity of these peptides to assume an ordered structure was investigated upon by changing the solvent's polarity and the temperature. A reduction of solvent polarity led to a significant increase in the α-helix content in the case of HR1, whereas only a slight change in the secondary structure was observed in the case of HR2. In both cases the conformational change followed a two-state transition model. The interaction of the peptides was monitored by the conformational change in the mixture with respect to the single peptides. However, formation of the complex did not significantly enhance thermal stability. A reliable estimation of the secondary structure was obtained by optimising the experimental conditions to collect CD data down to 180 nm, and by comparing the structure data yielded by different software packages.

Keywords: Circular dichroism; Secondary structure estimation; Peptide-peptide complexes; Thermal denaturation; Peptide folding

Effects of post-translational modifications on prion protein aggregation and the propagation of scrapie-like characteristics in vitro by Denise V. Dear; Duncan S. Young; Jurate Kazlauskaite; Filip Meersman; David Oxley; Judith Webster; Teresa J.T. Pinheiro; Andrew C. Gill; Igor Bronstein; Christopher R. Lowe (pp. 792-802).

Prion diseases, or transmissible spongiform encephalopathies (TSEs) are typically characterised by CNS accumulation of PrP^Sc, an aberrant conformer of a normal cellular protein PrP^C. It is thought PrP^Sc is itself infectious and the causative agent of such diseases. To date, no chemical modifications of PrP^Sc, or a sub-population thereof, have been reported. In this study we have investigated whether chemical modification of amino acids within PrP might cause this protein to exhibit aberrant properties and whether these properties can be propagated onto unmodified prion protein. Of particular interest were post-translational modifications resulting from physiological conditions shown to be associated with TSE disease. Here we report that in vitro exposure of recombinant PrP to conditions that imitate the end effects of oxidative/nitrative stress in TSE-infected mouse brains cause the protein to adopt many of the physical characteristics of PrP^Sc. Most interestingly, these properties could be propagated onto unmodified PrP protein when the modified protein was used as a template. These data suggest that post-translational modifications of PrP might contribute to the initiation and/or propagation of prion protein-associated plaques in vivo during prion disease, thereby high-lighting novel biochemical pathways as possible therapeutic targets for these conditions.

Keywords: Abbreviations; CNS; central nervous system; PrP; ^Sc; disease-associated isoform of prion protein; PrP; ^C; normal cellular isoform of PrP; TSE; transmissible spongiform encephalopathy; rPrP; recombinant full-length alpha-helical prion protein; MPO; myeloperoxidase; HPLC; high pressure liquid chromatography; FT-IR; Fourier transform infra-red spectroscopy; LC-MS/MS; liquid chromatography tandem mass spectrometry; ThT; thioflavine T; PK; proteinase K; PMSF; phenymethyl sulphonyl fluoride; NT; nitrotyrosine; MNP; myeloperoxidase, nitrite and peroxide reactants; NMR; nuclear magnetic resonance; SAFs; scrapie-associated fibrils; CJD; Creutzfeldt–Jakob Disease; PTM; post-translational modificationPrion protein; Aggregation; Post-translational modification; Nitro-tyrosine; Oxidative and nitrative stress

Reconstitution of apoglucose oxidase with FAD conjugates for biosensoring of progesterone by Geertruida A. Posthuma-Trumpie; Willy A.M. van den Berg; Dirk F.M. van de Wiel; Wim M.M. Schaaper; Jakob Korf; Willem J.H. van Berkel (pp. 803-812).

The reconstitution of Aspergillus niger apoglucose oxidase (apoGOx) with FAD conjugates for biosensoring of progesterone was investigated. ApoGOx prepared by partial unfolding of the protein under acidic conditions consisted of reconstitutable monomers (50±10%), reconstitutable dimers (20±10%) and irreversibly aggregated oligomers (30±20%). Incubation of monomeric apoGOx with FAD or N⁶-(6-aminohexyl)-FAD (ahFAD) restored glucose oxidase (GOx) activity and induced dimerization with stoichiometric incorporation of FAD. N⁶-(6-aminohexyl)-FAD progesterone conjugates also induced dimerization. However, holoenzyme reconstitution required relatively high concentrations of apoprotein and was dependent on the type of conjugate. Restoration to 25–50% of the original enzyme activity was obtained. Binding of the FAD-progesterone conjugates might hinder the closure of a protein lid needed for dimer formation. Our results illustrate the prospects of FAD conjugates in sensitive detection of progesterone in biological matrices in a biosensor based on the recombination of apoGOx with progesterone-conjugated FAD.

Keywords: Apoprotein; FAD; Biosensor; Glucose oxidase; Progesterone; Immunoassay

Pressure effects on the structure and function of human thioredoxin by Kazuyoshi Ado; Yoshihiro Taniguchi (pp. 813-821).

Thioredoxin is one of the major proteins that catalyze disulfide reduction and defines the thioredoxin superfamily bearing the CXXC structural motif. Human thioredoxin contains only 1 Trp residue proximal to the active site (WCGPC). We are interested in thioredoxin structure–function relationships, in particular, active site hydration and flexibility. Hence, in this study, we used hydrostatic pressure as a perturbation and monitored the conformational changes around the active site of thioredoxin by analyzing Trp fluorescence. The structure of thioredoxin was drastically altered by increasing pressure and did not completely refold after pressure release. The conformation in the active site vicinity was modified at low pressure (less than 100 MPa) and the Trp residue was completely exposed to aqueous medium at pressures above 350 MPa. Upon pressure release, thioredoxin showed no activity, although it folded 80% of the α-helical content relative to the native state. According to these results, pressure denaturation induces critical damage for the activity of thioredoxin, indicating extreme fragility of the active site with respect to pressure. This result is in contrast to the pressure effect on protein disulfide isomerase (PDI) which is organized by four thioredoxin-like domains including two WCGHC motifs.

Keywords: Abbreviations; PDI; Protein disulfide isomerase; Trx; Thioredoxin; TrxR; Thioredoxin reductase; CD; Circular dichroism; GR; Glutathione reductase; GSH; Glutathione reduced form; GSSG; Glutathione oxidized form; NADPH; β-Nicotinamide adenine dinucleotide phosphate; GdnHCl; Guanidine hydrochloride; CSM; Center of spectral massThioredoxin; High pressure; Tryptophan fluorescence; Reduction activity

The inhibition kinetics and thermodynamic changes of tyrosinase via the zinc ion by Hong-Yan Han; He-Chang Zou; Jun-Yeong Jeon; Ye-Jing Wang; Wei-An Xu; Jun-Mo Yang; Yong-Doo Park (pp. 822-827).

We found that Zn²⁺ conspicuously inactivated tyrosinase in a mixed-type inhibition manner: the final level of residual activity was abolished at the equilibrium state with concentration of 0.25 mM Zn²⁺. Changes of both K_m and V_max by various concentrations of Zn²⁺ in Lineweaver–Burk plot were observed. To see whether Zn²⁺ also induced conformational change of tyrosinase and how thermodynamical changes by ligand binding were occurred, the intrinsic fluorescence studies as well as calorimetric measurements were conducted. The results showed that the Zn²⁺ binding to tyrosinase directly induced conformational change of tyrosinase, and the changes of thermodynamic parameters such as enthalpy (Δ H), Gibbs free-energy (Δ G), and entropy (Δ S) were obtained as 60±7.0 kJ/mol, −14.54 kJ/mol and 248.53 J/(K mol), respectively. The inactivating effect of Zn²⁺ on tyrosinase was completely prevented by incubation with bovine serum albumin, which has a Zn²⁺ binding motif in its structure. We suggested that Zn²⁺ ligand-binding affected the substrate's accessibility due to the conformational changes and thus, the complex type of inhibition has occurred with the calorimetric changes.

Keywords: Abbreviations; ANS; 1-anilinonaphthalene-8-sulfonate; BPγG; bovine plasma gamma globulin; BSA; bovine serum albumin; DOPA; 3,4-dihydroxyphenylalanine; MES; 2-[N-morpholino]ethanesulfonic acidBovine plasma gamma globulin; Calorimetrics; Fluorescence; Melanogenesis; Metal binding; Tyrosinase

Biochemical and functional characterization of UDP-galactose 4-epimerase from Aeromonas hydrophila by Shivani Agarwal; Keshav Gopal; Tanuja Upadhyaya; Aparna Dixit (pp. 828-837).

Bacteria of genus Aeromonas, responsible for a variety of pathological conditions in humans and fish, are ubiquitous waterborne bacteria. Aeromonas produces several virulent factors including a complex of lipopolysaccharide and surface array protein, involved in colonization. UDP-galactose 4-epimerase (GalE) catalyzes the production of UDP-galactose, a precursor for lipopolysaccharide biosynthesis, and thus is an important drug target. GalE exhibits interspecies variation and heterogeneity at its structural and functional level and therefore, the differences between the GalE of the host and the pathogen can be exploited for drug designing. In the present study, we report biochemical and functional characterization of the recombinant GalE of Aeromonas hydrophila. Unlike GalE reported from all other species, the purified recombinant GalE of A. hydrophila was found to exist as a monomer. This is the first report of UDP-galactose 4-epimerase from any species being a monomer. The molecular mass of the 6×His-rGalE was determined to be 38271.477 ( m/ z). The 6×His-rGalE with a K_m of 0.5 mM for UDP-galactose exhibited optimum activity at 37 °C and pH 8–9. Spectrofluorimetric and CD analysis confirmed that the thermal inactivation was due to structural changes and not due to the NAD-dissociation. A relatively more ordered structure of the enzyme at pH 8 and 9 as compared to that at pH 6 or 7 suggests a key role of the electrostatic interactions in maintaining its native tertiary structure.

Keywords: Abbreviations; IPTG; isopropyl β-; d; -thiogalactopyranoside; GalE; UDP-galactose 4-epimerase; 6×His-rGalE; 6×-histidine-tagged recombinant UDP-galactose 4-epimerase; rGalE; recombinant UDP-galactose 4-epimerase without any tag; LPS; lipopolysaccharide; ANS; 1-anilinonapthalene-8-sulfonate; RFI (A.U.); Relative Fluorescence Intensity in Arbitrary Units Aeromonas hydrophila; UDP-galactose 4-epimerase; Circular dichroism; Fluorescence spectroscopy

Protein–protein interactions and disease: Use of S. cerevisiae as a model system by Wei-Tse Hsu; Chi Nam Ignatius Pang; Josefa Sheetal; Marc R. Wilkins (pp. 838-847).

Disease-causing mutations are increasingly being studied to see if they cause the loss or gain of protein–protein interactions. Because the interaction network of humans is poorly understood and difficult to investigate, here we propose the use of Saccharomyces cerevisiae as a model system for understanding the impact of disease-causing mutations on protein–protein interactions. Alignments of human disease-associated proteins and 379 yeast orthologs showed that 124 of these proteins have >40% sequence identity, with some orthologs having up to 89% identity. A total of 1826 amino acid mutations associated with human disease were found to map to invariant amino acids in yeast. These mutations were proportionately more likely to be non-conservative than non-disease associated polymorphisms for the same proteins ( p=0.016). Importantly, 73 of the mutations mapped to protein–protein interaction domains, implying a direct link between mutation and changes in protein interactivity. In the manuscript, all alignment information and tables that map mutations and diseases to yeast orthologs are given. This will help researchers experimentally test the impact of mutations on protein–protein interactions in S. cerevisiae and, by homology, explore the role of such mutations in the genesis of human disease.

Keywords: Abbreviations; 2-DE; two-dimensional polyacrylamide gel electrophoresis; LC-MS-MS; liquid chromatography tandem mass spectrometry; TAP; tandem affinity purification; OMIM; Online Mendelian Inheritance in Man; UniProt; universal protein resource; dbSNP; single nucleotide polymorphism database; SGD; Saccharomyces genome database; NDK; nucleoside diphosphate kinase; dRTA; distal renal tubular acidosis; NF1; neurofibromin; IRA2; inhibitory regulator protein; RASK; GTPase KRas; MSH2; DNA mismatch repair proteinProtein–protein interactions; Disease-associated mutations; Disease orthologs; Protein interaction domains; Single nucleotide polymorphisms; Saccharomyces cerevisiae

Analysis of peroxiredoxin decreasing oxidative stress in hypertensive aortic smooth muscle by Chang-Kwon Lee; Hyo Jin Kim; Youn Ri Lee; Hyeon Ha So; Hyo-Jun Park; Kyung-Jong Won; Taekyu Park; Kyung-Yung Lee; Hwan Myung Lee; Bokyung Kim (pp. 848-855).

To determine the role of peroxiredoxin (Prx) in response to oxidative stress and during hypertension in the vasculature, we identified Prx proteins and analyzed their antioxidant effects. Rat aortic smooth muscle contains all six Prxs (I–VI). Prx I, II, and VI shifted to its acidic site on two-dimensional polyacrylamide gel electrophoresis after exposure to H₂O₂. The total expression of Prx I and VI was increased in response to H₂O₂. The expression of Prx I, but not that of Prx II and VI, increases and the acidic form of Prx I and the sulfonic acid form of Prx (SO₃H-Prx) are more strongly expressed in the aortic smooth muscle of hypertensive rats than in that of normotensive control rats. Prxs were also found in the mesenteric artery, heart, and kidney. The expression levels of Prx I and VI were increased in mesenteric artery, but not heart and kidney, from hypertensive rats compared with that from normotensive rats. These results suggest that Prxs play a crucial role against oxidative stress in vascular smooth muscles during hypertension.

Keywords: Abbreviations; 2-DE; two-dimensional polyacrylamide gel electrophoresis; DOCA; deoxycorticosterone acetate; ERK; extracellular signal-regulated kinase; GPx; glutathione peroxidase; H; ₂; O; ₂; hydrogen peroxide; JNK; stress-activated protein kinase/c-; Jun; N-terminal kinase; MAPK; mitogen-activated protein kinase; PMSF; phenylmethylsulfonylfluoride; PSS; physiological salt solution; Prx; peroxiredoxin; ROS; reactive oxygen species; SD; Sprague–Dawley; SDS; sodium dodecyl sulphate; SHR; spontaneously hypertensive rats; WKY; Wistar Kyoto ratsPeroxiredoxin; Oxidative stress; Hypertension; Vascular smooth muscle; Sulfonic acid

Purification and partial characterization of N-hydroxy-l-phenylalanine decarboxylase/oxidase from Bacillus sp. strain OxB-1, an enzyme involved in aldoxime biosynthesis in the “aldoxime–nitrile pathway” by Yasuo Kato; Takashi Tsuda; Yasuhisa Asano (pp. 856-865).

An enzyme that catalyzes the conversion of N-hydroxy-l-phenylalanine to phenylacetaldoxime was shown to be present in the Z-phenylacetaldoxime-degrading bacterium, Bacillus sp. strain OxB-1. The aldoxime-forming enzyme, which is induced byl-phenylalanine, was purified 8,050-fold to apparent homogeneity with a yield of 15.2%. The enzyme has a subunit M_r of about 86,000. The enzyme converts N-hydroxy-l-phenylalanine ( K_m 0.99 mM) to only one geometrical isomer, namely Z-phenylacetaldoxime. Relatively large amounts of pyridoxal 5′-phosphate (PLP) are required to be present in the reaction mixture because PLP reacts non-enzymatically with the N-hydroxy amino acid substrate to form a nitrone. Several characteristics of the enzyme were compared with those of other PLP-dependent aromatic amino acid-converting enzymes described in the literature. The enzyme is tentatively named “ N-hydroxy-l-phenylalanine decarboxylase/oxidase”. Finally, the possible biosynthesis and metabolism of phenylacetaldoxime in Bacillus sp. strain OxB-1 is discussed.

Keywords: Abbreviations; NHP; N; -hydroxy-phenylalanine; PAOx; phenylacetaldoxime; Oxd; aldoxime dehydratase; NHase; nitrile hydratase; Nit; nitrilase; SCB; NaHCO; ₃; -Na; ₂; CO; ₃; buffer; KPB; potassium phosphate buffer; PLP; pyridoxal 5′-phosphateAldoxime; Nitrile; Microorganism; N; -Hydroxyamino acid

FTICR-MS analysis of 14-3-3 isoform substrate selection by Helene L. Cardasis; Paul C. Sehnke; Beth Laughner; John R. Eyler; David H. Powell; Robert J. Ferl (pp. 866-873).

The 14-3-3s are a ubiquitous class of eukaryotic proteins that participate in a second regulatory step in many phosphorylation-based signal transduction systems. The Arabidopsis family of 14-3-3 proteins represents a rather large 14-3-3 gene family. The biological motive for such diversity within a single protein family is not yet completely understood. The work presented here utilizes 14-3-3 micro-affinity chromatography in conjunction with Fourier transform ion cyclotron resonance mass spectrometry to survey the substrate sequence selectivity of two Arabidopsis 14-3-3 isoforms that represent the two major subclasses of this protein family. A method was developed to compare the relative binding of eight synthetic phosphopeptide sequences. The degree to which each phosphopeptide bound to either isoform was assigned a relative value, defined here as the binding ratio. The method provided a simple means for visualizing differences in substrate sequence selection among different 14-3-3 isoforms. A reproducible preference for specific phosphopeptide sequences was measured for both isoforms. This binding preference was consistent among the two classes of isoforms, suggesting that any pressure for isoform selectivity must reside outside the central core that interacts with the phosphopeptide sequence of the client.

Keywords: 14-3-3s; FTICR-MS; Phosphopeptide binding; Arabidopsis; Microaffinity chromatography

Stability of highly purified human paraoxonase (PON1): Association with human phosphate binding protein (HPBP) is essential for preserving its active conformation(s) by Daniel Rochu; Frédérique Renault; Cécile Cléry-Barraud; Eric Chabrière; Patrick Masson (pp. 874-883).

The biological role of human paraoxonase (PON1) remains unclear, whilst there is a consensus that the enzyme has a protective influence. A toxicological role, protecting from environmental poisoning by organophosphate derivatives drove earlier works, and more recently, clinical interest has focused on a protective role in vascular disease. PON1 resides essentially on HDL particles, a complex and dynamic molecular environment. Our recent discovery of the human phosphate binding protein (HPBP), displaying a firm propensity to associate with PON1, has steered new directions for characterizing PON1 functional state. Here, we report investigations on the effect of HPBP on oligomerization, storage and thermal stability of PON1. We found that purified PON1 is as a mixture of at least two states, and that the absence of HPBP favors homo-oligomerization of PON1 into state(s) of higher molecular size. We showed that HPBP allows stabilizing active conformation(s) of PON1 disencumbered of its natural environment. We also showed that PON1 exhibits intrinsically a remarkable thermal stability, and that the association of HPBP strongly contributes to slow the denaturation rate. A hybrid recombinant PON1 was shown more thermostable than the human enzyme, and its stability was unaffected by the presence of HPBP. Altogether, the results strongly encourage further study of the human enzyme.

Keywords: Abbreviations; CE; capillary electrophoresis; CWA; chemical warfare agent; DSC; differential scanning calorimetry; EOF; electro-osmotic flow; HDL; high density lipoprotein; HPBP; human phosphate binding protein; PON1; paraoxonase-1; OP; organophosphorus compounds; OPH; OP-hydrolase; rPON1; recombinant PON1Paraoxonase; Protein stability; Catalytic scavenger; Enzyme therapy; Nerve agent; Biopharmaceutical

Activation of DNA-hydrolyzing antibodies from the sera of autoimmune-prone MRL-lpr/lpr mice by different metal ions by Irina A. Kuznetsova; Irina A. Orlovskaya; Valentina N. Buneva; Georgy A. Nevinsky (pp. 884-896).

We have shown previously that electrophoretically and immunologically homogeneous polyclonal IgGs from the sera of autoimmune-prone MRL mice possess DNase activity. Here we have analyzed for the first time activation of DNase antibodies (Abs) by different metal ions. Polyclonal DNase IgGs were not active in the presence of EDTA or after Abs dialysis against EDTA, but could be activated by several externally added metal (Me²⁺) ions, with the level of activity decreasing in the order Mn²⁺≥Mg²⁺>Ca²⁺≥Cu²⁺>Co²⁺≥Ni²⁺≥Zn²⁺, whereas Fe²⁺ did not stimulate hydrolysis of supercoiled plasmid DNA (scDNA) by the Abs. The dependencies of the initial rate on the concentration of different Me²⁺ ions were generally bell-shaped, demonstrating one to four maxima at different concentrations of Me²⁺ ions in the 0.1–12 mM range, depending on the particular metal ion. In the presence of all Me²⁺ ions, IgGs pre-dialyzed against EDTA produced only the relaxed form of scDNA and then sequence-independent hydrolysis of relaxed DNA followed. Addition of Cu²⁺, Zn²⁺, or Ca²⁺ inhibited the Mg²⁺-dependent hydrolysis of scDNA, while Ni²⁺, Co²⁺, and Mn²⁺ activated this reaction. The Mn²⁺-dependent hydrolysis of scDNA was activated by Ca²⁺, Ni²⁺, Co²⁺, and Mg²⁺ ions but was inhibited by Cu²⁺ and Zn²⁺. After addition of the second metal ion, only in the case of Mg²⁺ and Ca²⁺ or Mn²⁺ ions an accumulation of linear DNA (single strand breaks closely spaced in the opposite strands of DNA) was observed. Affinity chromatography on DNA-cellulose separated DNase IgGs into many subfractions with various affinities to DNA and very different levels of the relative activity (0–100%) in the presence of Mn²⁺, Ca²⁺, and Mg²⁺ ions. In contrast to all human DNases having a single pH optimum, mouse DNase IgGs demonstrated several pronounced pH optima between 4.5 and 9.5 and these dependencies were different in the presence of Mn²⁺, Ca²⁺, and Mg²⁺ ions. These findings demonstrate a diversity of the ability of IgG to function at different pH and to be activated by different optimal metal cofactors. Possible reasons for the diversity of polyclonal mouse abzymes are discussed.

Keywords: Abbreviations; Abs; antibodies; Abzs; abzymes or catalytically active antibodies; AI; autoimmune; AID; autoimmune disease; MRL; MRL-lpr/lpr mice; SLE; systemic lupus erythematosus; SDS-PAGE; SDS- polyacrylamide gel electrophoresis; RDA; relative DNase activity; sc; supercoiled; VIP; vasoactive intestinal peptideAbzymes of MRL-lpr/lpr mice; DNA hydrolysis; Activation by metal ions

Comparative profiling of human saliva by intact protein LC/ESI-TOF mass spectrometry by Kevin M. Millea; Ira S. Krull; Asish B. Chakraborty; John C. Gebler; Scott J. Berger (pp. 897-906).

Human saliva is finding increasing interest for proteomic and biomarker-discovery studies, due to the ease of collection and potential for simpler processing workflows compared to serum or plasma. However, it is known that salivary protein composition can vary with physiological and environmental factors. In this work, we have examined intra- and inter-person variability of saliva protein composition using an LC/MS methodology to profile low molecular weight human salivary proteins. Whole saliva was analyzed from four individuals over three consecutive days. Additional samples were used to determine baseline analytical and sample processing variation and to identify phosphoproteins. Individuals were observed to have a similar salivary protein pattern over multiple days, although the expression levels of particular proteins were variable. Significant differences in protein profiles were observed between subjects, allowing for delineation of individuals based on their protein profile. Comparison with alkaline phosphatase treated saliva revealed that several identified proteins were singly, doubly, or triply phosphorylated.

Keywords: Saliva; Salivary fluid; Oral fluid; Intact protein; LC/MS; Top-Down; Protein modifications; Protein processing; Phosphorylation

Napin from Brassica juncea: Thermodynamic and structural analysis of stability by T.C. Jyothi; Sharmistha Sinha; Sridevi A. Singh; A. Surolia; A.G. Appu Rao (pp. 907-919).

The napin from Brassica juncea, oriental mustard, is highly thermostable, proteolysis resistant and allergenic in nature. It consists of two subunits – one small (29 amino acid residues) and one large (86 amino acids residues) – held together by disulfide bonds. The thermal unfolding of napin has been followed by differential scanning calorimetry (DSC) and circular dichroism (CD) measurements. The thermal unfolding is characterized by a three state transition with T_M1 and T_M2 at 323.5 K and 335.8 K, respectively; Δ C_P1 and Δ C_P2 are 2.05 kcal mol−¹ K−¹ and 1.40 kcal mol−¹ K−¹, respectively. In the temperature range 310–318 K, the molecule undergoes dimerisation. Isothermal equilibrium unfolding by guanidinium hydrochloride also follows a three state transition, N⇆ I⇆ U with Δ G_1H2O and Δ G_2H2O values of 5.2 kcal mol−¹ and 5.1 kcal mol−¹ at 300 K, respectively. Excess heat capacity values obtained, are similar to those obtained from DSC measurements. There is an increase in hydrodynamic radius from 20 Å to 35.0 Å due to unfolding by guanidinium hydrochloride. In silico alignment of sequences of napin has revealed that the internal repeats (40%) spanning residues 31 to 60 and 73 to 109 are conserved in all Brassica species. The internal repeats may contribute to the greater stability of napin. A thorough understanding of the structure and stability of these proteins is essential before they can be exploited for genetic improvements for nutrition.

Keywords: Abbreviations; T; _M; transition temperature; Δ; H; _C; calorimetric enthalpy; Δ; H; _V; van't Hoff's enthalpy; Δ; C; _P; excess heat capacity; DSC; differential scanning calorimetry; CD; circular dichroism; PVDF; polyvinyl difluoride; GdnHCl; guanidine hydrochloride; TNBS; trinitrobenzene sulfonic acid; DTT; dithiothreitol; TPCK; tosyl phenylalanine chloromethyl ketone; TLCK; tosyl lysine chloro methyl ketone; EDC; [1-ethyl-3-(3-dimethyl amino propyl) carbodiimide]; ANS; 8-aniline naphthalene sulfonic acid; TFA; trifluoroacetic acid; ASA; accessible surface areaNapin; Thermal stability; Brassica juncea; Association; Protein unfolding; Internal repeats

Dual polarisation interferometry analysis of copper binding to the prion protein: Evidence for two folding states by Andrew R. Thompsett; David R. Brown (pp. 920-927).

The prion protein is a copper binding glycoprotein expressed in neurones and other cells. Conversion of this protein to an abnormal isoform is central to the cause of prion diseases or transmissible spongiform encephalopathies. Detecting slight structural differences between different forms of the prion protein could be essential to understanding the role of the protein in health and disease. Dual polarisation interferometry (DPI) is a new method that allows detection of small structural differences. We used this technique to evaluate the effectiveness of DPI in the analysis of metal binding to recombinant mouse prion protein. DPI was able to measure mass change in the prion protein following addition of copper and could identify reproducible differences in the structure of prion protein dependent on how metal was added to the protein. These slight structural differences were confirmed by the use of circular dichroism spectroscopy and Fourier-transformed infra-red spectroscopy. These results suggest that DPI can provide important information on both transitory and stable structural difference that are induced in the prion protein. This technique could be important not only for the study of metal–protein interactions but also small structural differences that could define prion strains.

Keywords: Copper; Prion; Metal; Scrapie; Dual polarisation interferometry

Hydration state change of proteins upon unfolding in sugar solutions by Osato Miyawaki (pp. 928-935).

Change in hydration number of proteins upon unfolding, Δ n, was obtained from the analysis of thermal unfolding behavior of proteins in various sugar solutions with water activity, a_W, varied. By applying the reciprocal form of Wyman–Tanford equation, Δ n was determined to be 133.9, 124.1, and 139.2 per protein molecule for ribonuclease A at pH=5.5, 4.2, and 2.8, respectively, 201.4 for lysozyme at pH=5.5, and 100.1 for α-chymotripnogen A at pH=2.0. Among the sugars tested, reducing sugars gave the lower apparent Δ n as compared with nonreducing sugars probably because of the direct interaction of reducing terminal with amino group of proteins at a high temperature. From the knowledge of Δ n, a new thermodynamic model for protein stability was proposed with explicit consideration for hydration state change of protein upon unfolding. From this model, the contribution of a_W was proven to be always positive for stabilization of proteins and its effect is not negligible depending onΔ n and a_W.

Keywords: Thermal unfolding of protein; Hydration number; Water activity; Free energy for protein stability; Ribonuclease A

Plasmodium falciparum dUTPase: Studies on protein stability and binding of deoxyuridine derivatives by Indalecio Quesada-Soriano; Juan Alexander Musso-Buendia; Ramiro Tellez-Sanz; Luis Miguel Ruíz-Pérez; Carmen Barón; Dolores González-Pacanowska; Luis García-Fuentes (pp. 936-945).

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase), a ubiquitous enzyme preventing a deleterious incorporation of uracil into DNA, has been thought of as a novel target for anticancer and antiviral drug design. The interaction of Plasmodium falciparum dUTPase (PfdUTPase) with deoxyuridine derivatives (dU, dUMP, dUDP and dUpNHpp) has been studied thermodynamically by both isothermal titration and differential scanning calorimetry. ITC shows no cooperativity for the binding of these derivatives. Dependencies in the binding thermodynamic parameters (enthalpy, entropy and Gibbs energy changes) with the number of phosphate groups in the nucleotide are obtained, and from the heat capacity changes no significant conformational changes upon binding are inferred. DSC shows PfdUTPase trimer is very stable but denatures irreversibly, with a more complex denaturation profile than other homologous trimeric dUTPases. The presence of magnesium ions does not influence the denaturation profile, while the presence of deoxyuridine derivatives increases the stability. The increase depends upon nucleotide concentration and type, with dUDP having the greater effect.

Keywords: Abbreviations; dUTPase; dUTP pyrophosphatase (EC 3.6.1.23); α, β-imido-dUTP (or dUpNHpp); 2′-deoxyuridine 5′-(α,β-imido)triphosphate; ITC; isothermal titration calorimetry; DSC; differential scanning calorimetryCalorimetry; dUTPase; Plasmodium falciparum; Thermal denaturation; Binding

Serum albumins have five sites for binding of cationic dendrimers by Dzmitry Shcharbin; Magdalena Janicka; Michal Wasiak; Bartlomiej Palecz; Magdalena Przybyszewska; Marian Zaborski; Maria Bryszewska (pp. 946-951).

The detailed analysis of the interaction between PAMAM G4 dendrimer and serum albumins was performed using circular dichroism, isothermal titration calorimetry, capillary electrophoresis, zeta-potential and fluorescence polarization. It was shown that serum albumins and PAMAM G4 dendrimer form the complex with stoichiometry of 4–6:1 for G4:HSA and 4–5:1 for G4:BSA molar ratio. The possible sites of PAMAM G4 dendrimers binding to protein surface were discussed. Also, it has been proposed that dendrimer does not significantly affect the protein secondary structure studied by circular dichroism.

Keywords: PAMAM dendrimer; Serum albumin; Complex; Binding sites

Two distinct sites of interaction form the calponin: gelsolin complex and two calcium switches control its activity by Imen Ferjani; Abdellatif Fattoum; Sutherland K. Maciver; Mohamed Manai; Yves Benyamin; Claude Roustan (pp. 952-958).

Gelsolin and calponin are well characterized actin-binding proteins that form a tight gelsolin:calponin complex (GCC). We show here that the GCC is formed through two distinct interfaces. One of these is formed between 144–182 of calponin and 25–150 of gelsolin (G1). The second is a calcium-sensitive site centred on calponin's CH domain, and the C-terminal half of gelsolin (G4–6). The behaviour of this second interface is dependent on the presence of calcium and so it is possible that potential GCC-binding partners may be selected by calcium availability. Actin is one such GCC-binding partner and we show that a larger complex is formed with monomeric actin in calcium. The stoichiometry of this complex is determined to be 1 gelsolin/1 calponin/2 G-actins (GCA₂). Both actin monomers bind the GCC through gelsolin. Both calponin and gelsolin are reported to play signaling roles in addition to their better-characterized actin-binding properties and it is possible that the GCC regulates both of these functions.

Keywords: Abbreviations; CH; calponin homology domain; G1–6; the six repeated domains that comprise gelsolin; NBD; 7-chloro-4-nitrobenzeno-2-oxa-1,3-diazole; 1,5-AEDANS; N; -iodoacetyl-; N′; -(sulpho-1-naphthyl)-ethylenediamineActin; Cytoskeleton; Cell signalling; Actin-binding proteins

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BBA - Proteins and Proteomics (v.1774, #7)

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BBA - Proteins and Proteomics (v.1774, #7)