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BBA - Proteins and Proteomics (v.1750, #1)

Editorial Board (pp. ii).

Pressure-tuning FT-IR spectroscopic study on the helix–coil transition of Ala-rich oligopeptide in aqueous solution by Takahiro Takekiyo; Akio Shimizu; Minoru Kato; Yoshihiro Taniguchi (pp. 1-4).
We investigated the effect of pressure on the helix–coil transition of an Ala-rich peptide (AK16: YGAAKAAAAKAAAAKA-NH2) in aqueous solution by FT-IR spectroscopy. The spectra of the amide I' region of AK16 in aqueous solution was decomposed into some component bands using a curve fitting method. The peak at around 1635 cm−1 corresponding to the solvent exposed α-helix conformer increases with increasing pressures, while the peak at around 1655 cm−1 corresponding to the random coil conformer decreases. From the pressure dependence of the band intensities, we determined the volume change from the α-helix to random coil conformers of AK16 to be +10.5±0.3 cm3/mol. The positive volume change is different from the negative volume change generally observed in the pressure denaturation of proteins.

Keywords: Ala-rich peptide; Pressure effect; Helix–coil transition; Partial molar volume, FT-IR spectroscopy


Crystallization and preliminary crystallographic analysis of Bacillus thuringiensis AHL-lactonase by Myung Hee Kim; Hye Ok Kang; Beom Sik Kang; Kyung-Jin Kim; Won-Chan Choi; Tae-Kwang Oh; Choong Hwan Lee; Jung-Kee Lee (pp. 5-8).
The quorum sensing (QS) systems in Gram-negative bacteria are mostly associated with diffusible N-acyl-l-homoserine lactones (AHLs). AHL-degrading enzymes hydrolyze the AHLs into inactive molecules, thereby blocking the QS systems that are closely linked to virulence factor production and biofilm formation. Consequently, these enzymes have recently attracted intense interest for the development of anti-infection therapies for plants and animals. However, despite significant progress in the investigation of AHL-degrading enzymes, no structure is yet available. Accordingly, this study reports on the expression and purification of the AHL-lactonase from Bacillus thuringiensis subsp. kurstaki HD263, as well as the successful crystallization of the enzyme. High-quality native crystals were obtained and a complete data set collected at 2.0 Å resolution. The native crystal was found to belong to the space group P212121, with unit cell parameters a=52.7 Å, b=55.9 Å, and c=74.1 Å and one molecule in the asymmetric unit. MAD data were also collected at 2.4 Å resolution for a SeMet-substituted crystal.

Keywords: Quorum sensing; AHL-lactonase; N; -acyl-; l; -homoserine lactone; Crystallization


Crystallization and preliminary X-ray diffraction analysis of importin-α complexed with NLS peptidomimetics by Marcos R.M. Fontes; Trazel Teh; Ryan D. Riell; Seung Bum Park; Robert F. Standaert; Bostjan Kobe (pp. 9-13).
Importin-α is the nuclear import receptor that recognizes cargo proteins with nuclear localization sequences (NLSs). The study of NLS peptidomimetics can provide a better understanding of the requirements for the molecular recognition of cargo proteins by importin-α, and potentially engender a large number of applications in medicine. Importin-α was crystallized with a set of six NLS peptidomimetics, and X-ray diffraction data were collected in the range 2.1–2.5 Å resolution. Preliminary electron density calculations show that the ligands are present in the crystals.

Keywords: Abbreviations; Impα; importin-α; NLS; nuclear localization sequence; Arm repeat; armadillo repeat; Boc; tert; -butoxycarbonylCrystallization; X-ray crystallography; Importin-α; Karyopherin-α; Nuclear localization sequence; NLS peptidomimetic ligand


Crystallization and preliminary X-ray analysis of the C-terminal WRKY domain of Arabidopsis thaliana WRKY1 transcription factor by Ming-Rui Duan; Hui Ren; Peng Mao; Chun-Hong Wei; Yu-He Liang; Yi Li; Xiao-Dong Su (pp. 14-16).
The C-terminal WRKY domain of Arabidopsis thaliana WRKY1 protein, a transcription factor, was cloned and expressed. The expressed protein was then purified and crystallized. The preliminary X-ray analysis was undertaken. The crystal diffracted to 2.50 Å resolution in-house and belongs to space group P21 with unit-cell parameters a=64.10 Å, b=34.88 Å, c=114.72 Å, β=90.49°.

Keywords: WRKY protein; Transcription factor; Crystallization; X-ray diffraction


Effect of dioxane on the structure and hydration–dehydration of α-chymotrypsin as measured by FTIR spectroscopy by Vladimir A. Sirotkin (pp. 17-29).
A new experimental approach based on FTIR spectroscopic measurements was proposed to study simultaneously the adsorption/desorption of water and organic solvent on solid enzyme and corresponding changes in the enzyme secondary structure in the water activity range from 0 to 1.0 at 25 °C. The effect of dioxane on the hydration/dehydration and structure of bovine pancreatic α-chymotrypsin (CT) was characterized by means of this approach. Dioxane sorption exhibits pronounced hysteresis. No sorbed dioxane was observed at low water activities ( aw<0.5) during hydration. At aw about 0.5, a sharp increase in the amount of sorbed dioxane was observed. Dioxane sorption isotherm obtained during dehydration resembles a smooth curve. In this case, CT binds about 150 mol dioxane/mol enzyme at the lowest water activities. Three different effects of dioxane on the water binding by the initially dried CT were observed. At aw<0.5, water adsorption is similar in the presence and absence of dioxane. It was concluded that the presence of dioxane has little effect on the interaction between enzyme and tightly bound water at low aw. At aw>0.5, dioxane increases the amount of water bound by CT during hydration. This behavior was interpreted as a dioxane-assisted effect on water binding. Upon dehydration at low water activities, dioxane decreases the water content at a given aw. This behavior suggests that the suppression in the uptake of water during dehydration may be due to a competition for water-binding sites on chymotrypsin by dioxane. Changes in the secondary structure of CT were determined from infrared spectra by analyzing the structure of amide I band. Dioxane induced a strong band at 1628 cm−1 that was assigned to the intermolecular β-sheet aggregation. Changes in the intensity of the 1628 cm−1 band agree well with changes in the dioxane sorption by CT. An explanation of the dioxane effect on the CT hydration and structure was provided on the basis of hypothesis on water-assisted disruption of polar contacts in the solid enzyme. The reported results demonstrate that the hydration and structure of α-chymotrypsin depend markedly on how enzyme has been hydrated — whether in the presence or in the absence of organic solvent. A qualitative model was proposed to classify the effect of hydration history on the enzyme activity- aw profiles.

Keywords: Bovine pancreatic α-chymotrypsin; Thin film; Enzyme hydration; Secondary structure; Organic solvent sorption; Hysteresis phenomenon; Fourier transform infrared spectroscopy


Role of the disulphide bridge in folding, stability and function of porcine odorant binding protein: Spectroscopic equilibrium studies on C63A/C155A double mutant by Mariella Parisi; Alberto Mazzini; Robert Tibor Sorbi; Roberto Ramoni; Stefano Grolli; Roberto Favilla (pp. 30-39).
Porcine odorant binding protein (pOBP) contains a single disulphide bridge linking residues Cys63 and Cys155. In order to get information on the role played by this crosslink in determining the structural and functional properties of the protein, we substituted these two Cys residues with two Ala residues by site directed mutagenesis and investigated the changes in folding, stability and functional features, as detected by fluorescence and circular dichroism measurements. In particular, we studied both chemical and thermal unfolding/refolding processes under equilibrium conditions, the first induced by guanidinium hydrochloride and the second by raising the temperature from 15 to 90 °C. Chemical unfolding curves, as obtained from intrinsic fluorescence and far-UV circular dichroism data, can be fitted by a simple two-state cooperative sigmoidal function; however, their partial overlap ( C1/2=0.57±0.05 from fluorescence and 0.66±0.03 from CD) suggests the formation of an intermediate, which lacks tertiary structural features. Thermal unfolding was found to be reversible if the protein was heated up to 65 °C, but irreversible above that temperature because of aggregation. The thermodynamic unfolding parameters of this double mutant protein, when compared to those of the wild type protein, clearly point out the important role played by the disulphide bridge on the stability and function of this protein family and probably of many other lipocalins.

Keywords: Abbreviations; AMA; 1-aminoanthracene; CD; circular dichroism; GdnHCl; guanidinium hydrochloride; OBP(s); odorant binding protein(s); wt pOBP; wild type porcine OBP; mutant pOBP; C63A/C155A mutant pOBP; bOBP; bovine OBP; TRIS; 20 mM Tris–HCl buffer pH 7.8Protein folding; Protein stability; Disulphide bridge; Odorant binding protein; Fluorescence and circular dichroism


Conserved protein TTHA1554 from Thermus thermophilus HB8 binds to glutamine synthetase and cystathionine β-lyase by Ryoichi Arai; Madoka Nishimoto; Mitsutoshi Toyama; Takaho Terada; Seiki Kuramitsu; Mikako Shirouzu; Shigeyuki Yokoyama (pp. 40-47).
TTHA1554 was found as a hypothetical protein composed of 95 amino acids in the genome of the extremely thermophilic bacterium, Thermus thermophilus HB8. Proteins homologous to TTHA1554 are conserved in several bacteria and archaea, although their functions are unknown. To investigate the function of TTHA1554, we identified interacting proteins by using a pull-down assay and mass spectrometry. TTHA1329, which is glutamine synthetase, and TTHA1620, a putative aminotransferase, were identified as TTHA1554 binding proteins. The interactions with TTHA1329 and TTHA1620 were validated using in vitro pull-down assays and surface plasmon resonance biosensor assays with recombinant proteins. Since sequence homology analyses suggested that TTHA1620 was a pyridoxal 5′-phosphate-dependent enzyme, such as an aminotransferase, a cystathionine β-lyase or a cystalysin, putative substrates were investigated. When cystathionine, cystine and S-methylcysteine were used as substrates, pyruvate was produced by TTHA1620. The data revealed that TTHA1620 has cystathionine β-lyase enzymatic activity. When TTHA1554 was added to the reaction mixtures, the glutamine synthetase and cystathionine β-lyase enzymatic activities both increased by approximately two-fold. These results indicated that TTHA1554 is a novel protein (we named it GCBP: glutamine synthetase and cystathionine β-lyase binding protein) that binds to glutamine synthetase and cystathionine β-lyase.

Keywords: Abbreviations; CBB; Coomassie Brilliant Blue; DTT; dithiothreitol; GCBP; glutamine synthetase and cystathionine β-lyase binding protein; MALDI-TOF; matrix-assisted laser desorption ionization-time of flight; NADH; reduced nicotinamide adenine dinucleotide; PAGE; polyacrylamide gel electrophoresis; PBS; phosphate buffered saline; PLP; pyridoxal 5′-phosphate; PMSF; phenylmethanesulfonyl fluoride; SDS; sodium dodecyl sulfate; SPR; surface plasmon resonanceCystathionine β-lyase; Glutamine synthetase and cystathionine β-lyase binding protein; Glutamine synthetase; Hypothetical protein; Pull-down assay; Thermophilic bacteria


NMR and mass spectrometry studies of putative interactions of cell cycle proteins pRb and CDK6 with cell differentiation proteins MyoD and ID-2 by Pawel Smialowski; Mahavir Singh; Aleksandra Mikolajka; Sudipta Majumdar; Joma K. Joy; Narasimharao Nalabothula; Marcin Krajewski; Roland Degenkolbe; Hans-Ulrich Bernard; Tad A. Holak (pp. 48-60).
Cell growth and differentiation require precise coordination of cell cycle and differentiation proteins. This can be achieved by direct interactions between proteins, by indirect interaction in multiprotein complexes, or by modulation of gene expression levels of partner proteins. Contradictory data abound in the literature regarding the binding between some central cell cycle proteins, pRb, and CDK6, with myogenic differentiation promoting, MyoD, and inhibiting, Id-2, factors. We have tested these interactions using pure proteins and in vitro biophysical and biochemical methods, which included mass spectrometry, nuclear magnetic resonance (NMR), the affinity chromatography pull-down assays, and gel filtration chromatography. Using this multimethod approach, we were able to document interactions between pRb and HPV-E7, pRb and SV40 large T antigen, CDK6 and p19, and MyoD and DNA. Using the same methods, we could unambiguously show that there is no direct protein–protein interaction in vitro between the small pocket domain of pRb and the bHLH domain of MyoD, the small pocket domain of pRb and Id-2, and CDK6 and a 15-amino-acid peptide from the C-terminal domain of MyoD. Indirect interactions, through additional binding partners in multiprotein complexes or modulation of gene expression levels of these proteins, are therefore their probable mode of action.

Keywords: MyoD; Id-2; Retinoblastoma protein; CDK6; Interaction; Complex


Deglycosylation of glucoamylase from Aspergillus niger: Effects on structure, activity and stability by Javad Jafari-Aghdam; Khosro Khajeh; Bijan Ranjbar; Mohsen Nemat-Gorgani (pp. 61-68).
A comparative structure–function study was performed to establish possible roles of carbohydrates in stabilization of glycoproteins, using glucoamylase (GA) as a model system. In addition to kinetic properties, stability toward elevated temperatures, extremes of pH, high salt concentrations together with circular dichroism, intrinsic/extrinsic fluorescence studies, proteolysis and affinity for interaction with hydrophobic ligands were investigated. Related to all the main properties examined, with one exception, glycosylation provided improvement in functional characteristics of the enzyme, especially in relation to its thermostability. Results are explained in terms of provision of stabilizing intermolecular interactions by the sugar molecules. The improvement in protein rigidity together with reduction of surface hydrophobicity appear to be especially important in relation to prevention of aggregation, an important mechanism of irreversible thermoinactivation, occurring at elevated temperatures.

Keywords: Glucoamylase; Irreversible thermoinactivation; Aggregation; Refolding; Deglycosylation


The interaction of mitochondrial translational initiation factor 2 with the small ribosomal subunit by Angela C. Spencer; Linda L. Spremulli (pp. 69-81).
Bovine mitochondrial translational initiation factor 2 (IF-2mt) is organized into four domains, an N-terminal domain, a central G-domain and two C-terminal domains. These domains correspond to domains III–VI in the six-domain model of Escherichia coli IF-2. Variants in IF-2mt were prepared and tested for their abilities to bind the small (28S) subunit of the mitochondrial ribosome. The binding of wild-type IF-2mt was strong ( Kd∼10–20 nM) and was not affected by fMet-tRNA. Deletion of the N-terminal domain substantially reduced the binding of IF-2mt to 28S subunits. However, the addition of fMet-tRNA stimulated the binding of this variant at least 2-fold demonstrating that contacts between fMet-tRNA and IF-2mt can stabilize the binding of this factor to 28S subunits. No binding was observed for IF-2mt variants lacking the G-domain which probably plays a critical role in organizing the structure of IF-2mt. IF-2mt contains a 37-amino acid insertion region between domains V and VI that is not found in the prokaryotic factors. Mutations in this region caused a significant reduction in the ability of the factor to promote initiation complex formation and to bind 28S subunits.

Keywords: Abbreviations; IF-2; mt; bovine mitochondrial initiation factor 2; fMet-tRNA; formylmethionyl-tRNA; PVDF; polyvinylidene fluoride; GDPNP; guanosine 5′-[β,γ-imido]triphosphate; SA-PMP; Streptavidin MagneSphere® Paramagnetic ParticlesBovine; Mitochondria; Protein synthesis; tRNA; Initiation; Initiation factor 2; Small ribosomal subunit


Disulfide-linked dimers of human adrenaline synthesizing enzyme PNMT are catalytically active by Christine L. Gee; Amanda Nourse; A-Yen Hsin; Qian Wu; Joel D. Tyndall; Gary L. Grunewald; Michael J. McLeish; Jennifer L. Martin (pp. 82-92).
The crystal structure of human phenylethanolamine N-methyltransferase (hPNMT) reveals a disulfide-linked dimer, despite the presence of reducing agent in the crystallisation conditions. By removing the reducing agent, hPNMT crystals grow more rapidly and at lower protein concentrations. However, it was unclear whether the disulfide bonds are only present in the crystal form or whether these affect enzyme activity. The solution oligomeric state of hPNMT was investigated using biochemical techniques and activity assays. We found that in the absence of reducing agent, hPNMT forms dimers in solution. Furthermore, the solution dimer of hPNMT incorporates disulfide bonds, since this form is sensitive to reducing agent. The C48A and C139A mutants of hPNMT, which are incapable of forming the disulfide bond observed in the crystal structure, have a decreased propensity to form dimer in solution. Those dimers that do form are also sensitive to reducing agent. Further, the C48A/C139A double mutant shows only monomeric behaviour. Both dimeric and monomeric hPNMT, as well as mutants have wildtype enzyme activity. These results show that a variety of disulfides, including those observed in the crystal structure, can form in solution. In addition, disulfide-linked dimers are as active as the monomeric enzyme indicating that the crystal structure of the protein is a valid target for inhibitor design.

Keywords: Abbreviations; AdoHcy; S-adenosyl-; l; -homocysteine; AdoMet; S-adenosyl-; l; -methionine; βME; β-mercaptoethanol; AUC; analytical ultracentrifugation; DTT; dithiothreitol; EDTA; ethylene-diamine tetraacetic acid; GSH; reduced glutathione; GSSG; oxidized glutathione; PAGE; polyacrylamide gel electrophoresis; SDS; sodium dodecyl sulfate; Native-PAGE; PAGE without addition of SDS; PEA; phenylethanolamine; PEG; polyethylene glycol; PNMT; Phenylethanolamine; N; -methyltransferase; hPNMT; human PNMT; hPNMT-His; C-terminal 6xHistidine-tagged hPNMT; C48A hPNMT; C48A mutant of hPNMT-His; C139A hPNMT; C139A mutant of hPNMT-His; C48A/C139A hPNMT; double mutant of C48A and C139A of hPNMT-His; rmsd; root mean square deviation; SEC; size exclusion chromatographyCatecholamine synthesis; Disulfide bond formation; Catalytic activity; Dimerisation; Chromatography; Analytical ultracentrifugation; Protein purification


Guanidine hydrochloride denaturation of human serum albumin originates by local unfolding of some stable loops in domain III by Basir Ahmad; Md Zulfazal Ahmed; Soghra Khatun Haq; Rizwan Hasan Khan (pp. 93-102).
The effect of guanidine hydrochloride (GnHCl) on the global stability of human serum albumin (HSA) has been studied by fluorescence and circular dichroism spectroscopic measurements. The differential stability of native conformation of three HSA domains were explored by using domain-specific ligands, hemin (domain I), chloroform (domain II), bilirubin (at domain I/domain II interface) and diazepam (domain III). GnHCl induced unfolding transition curves as monitored by probes for secondary and tertiary structures were cooperative but noncoincidental. A strong ANS binding to the protein was observed around 1.8 M GnHCl, suggesting existence of intermediate states in the unfolding pathway of HSA. A gradual decrease (in the GnHCl concentration range 0.0–1.8 M) in the binding of diazepam indicates that domain III is the most labile to GnHCl denaturation. A significant increase in the binding of bilirubin up to 1.4 M GnHCl and decrease thereafter leading to complete abolishment of bilirubin binding at around 2.0 M GnHCl suggest favorable rearrangement and separation of domains I and II at 1.4 and 2.0 M GnHCl concentration, respectively. Above 1.6 M GnHCl, decrease of the binding of hemin, a ligand for domain I, chloroform, which binds in domain II and lone tryptophanyl fluorescence (Trp-214 located in domain II) indicate that at higher concentration of GnHCl domains I and II start unfolding simultaneously but the stability of domain I (7.4 Kcal/mol) is much more than domain II (4.3 Kcal/mol). A pictorial model for the unfolding of HSA domains, consistent with all these results, has been formulated, suggesting that domain III is the most labile followed by domain II while domain I is the most stable. A molten globule like state of domain III around 1.8 M GnHCl has also been identified and characterized.

Keywords: Abbreviations; Dom; domain; F; D; fraction denatured; Fn; fraction native; GnHCl; Guanidine hydrochloride; HSA; Human serum albumin; I; intermediate state; N; native state; Trp; tryptophan; U; unfolded stateHuman serum albumin; Domain stability; Molten globule; Ligand binding; Circular dichroism
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