Forgot your password?
New user?
New Window Opens on the Secret Life of Microbes: Scientists Develop First Microbial Profiles of Ecosystems
CAS announces the release of SciFinder Scholar for Mac OS X
Press Releases
Press Releases
| Check out our New Publishers' Select for Free Articles |
BBA - Proteins and Proteomics (v.1764, #5)
In vitro refolded napin-like protein of Momordica charantia expressed in Escherichia coli displays properties of native napin by Aruna Vashishta; Tejram Sahu; Anshu Sharma; Shailesh Kumar Choudhary; Aparna Dixit (pp. 847-855).
Napins belong to the family of 2S albumin seed storage proteins and are shown to possess antifungal activity. Napins, in general, consist of two subunits (derived from single precursor) linked by disulphide bridges. Usually, reducing environment of the E. coli cytosol is not conducive for proper folding of heterodimeric proteins containing disulphide bridges. Present investigation reports for the first time expression of napin-like protein of Momordica charantia (rMcnapin) in E. coli and its in vitro refolding to produce biologically active protein. Full-length cDNA encoding napin-like protein (2S albumin) was isolated from M. charantia seeds by immunoscreening a cDNA expression library. The cDNA consisted of an open reading frame encoding a protein of 140 amino acid residues. The 36 amino acids at the N-terminus represent the signal and propeptide. The region encoding small and large chains of the M. charantia napin is separated by a linker of 8 amino acid residues. The region encoding napin (along with the linker) was PCR amplified, cloned into pQE-30 expression vector and expressed in E. coli. rMcnapin expressed as inclusion bodies was solubilized and purified by Ni2+-NTA affinity chromatography. The denatured and reduced rMcnapin was refolded by rapid dilution in an alkaline buffer containing glycerol and redox couple (GSH and GSSG). Refolded His-rMcnapin displayed similar spectroscopic properties as that of mature napin-like protein of M. charantia with 48.7% α-helical content. In addition, it also exhibited antifungal activity against T. hamatum with IC50 of 3 μg/ml. Refolded His-rMcnapin exhibited ∼90% antifungal activity when compared with that of mature napin-like protein of M. charantia. Thus, a heterologous expression system and in vitro refolding conditions to obtain biologically active napin-like protein of M. charantia were established.
Keywords: Abbreviations; IPTG; isopropyl-thio-β-; d; -galactopyranoside; pfu; plaque-forming units; His-rMcnapin; 6× histidine tagged recombinant napin of; Momordica charantia; IPF; internal processed fragment; GnHCl; Guanidium–HCl Momordica charantia; Napin-like protein cDNA; antifungal activity; in vitro refolding; recombinant napin
NMR studies of BPTI aggregation by using paramagnetic relaxation reagents by Andrea Bernini; Ottavia Spiga; Arianna Ciutti; Vincenzo Venditti; Filippo Prischi; Mariangela Governatori; Luisa Bracci; Barbara Lelli; Silvia Pileri; Mauro Botta; Alessandro Barge; Franco Laschi; Neri Niccolai (pp. 856-862).
Paramagnetic probes, whose approach to proteins can be monitored by nuclear magnetic resonance (NMR) studies, have been found of primary relevance for investigating protein surfaces accessibility. Here, paramagnetic probes are also suggested for a systematic investigation on protein aggregation. Bovin pancreatic trypsin inhibitor (BPTI) was used as a model system for aggregation by analyzing its interaction with TEMPOL and Gd(III)DTPA-BMA. Some of the measured paramagnetic relaxation rates of BPTI protons exhibited a reverse dependence on protein concentration, which can be attributed to the formation of transient BPTI aggregates.
Keywords: Abbreviations; BPTI; bovin pancreatic trypsin inhibitor; EPR; Electronic Paramagnetic Resonance; Gd(III)DTPA-BMA; Gd(III)-diethylenetriamine pentaacetic acid-bismethylamide; NMR; Nuclear Magnetic Resonance; SPR; Surface Plasmonic Resonance; TEMPOL; 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxylNMR; Paramagnetic probe; TEMPOL; Surface accessibility; Gd(III)DTPA-BMA; Protein aggregation; BPTI
Isolation and characterization of cotiaractivase, a novel low molecular weight prothrombin activator from the venom of Bothrops cotiara by Yotis A. Senis; Paul Y. Kim; Gemma L.J. Fuller; Ã?ngel GarcÃa; Sripadi Prabhakar; Mark C. Wilkinson; Helen Brittan; Nicole Zitzmann; Robin Wait; David A. Warrell; Steve P. Watson; Aura S. Kamiguti; R. David G. Theakston; Michael E. Nesheim; Gavin D. Laing (pp. 863-871).
In this study, we isolated a novel prothrombin activator from the venom of Bothrops cotiara, a Brazilian lance-headed pit viper (Cotiara, Jararaca preta, Biocotiara), which we have designated “cotiaractivase� (prefix: cotiar- from B. cotiara; suffix: -activase, from prothrombin activating activity). Cotiaractivase was purified using a phenyl-Superose hydrophobic interaction column followed by a Mono-Q anion exchange column. It is a single-chain polypeptide with a molecular weight of 22,931 Da as measured by mass spectroscopy. Cotiaractivase generated active α-thrombin from purified human prothrombin in a Ca2+-dependent manner as assessed by S2238 chromogenic substrate assay and SDS-PAGE. Cotiaractivase cleaved prothrombin at positions Arg271–Thr272 and Arg320–Ile321, which are also cleaved by factor Xa. However, the rate of thrombin generation by cotiaractivase was approximately 60-fold less than factor Xa alone and 17×106-fold less than the prothrombinase complex. The enzymatic activity of cotiaractivase was inhibited by the chelating agent EDTA, whereas the serine protease inhibitor PMSF had no effect on its activity, suggesting that it is a metalloproteinase. Interestingly, S2238 inhibited cotiaractivase activity non-competitively, suggesting that this toxin contains an exosite that allows it to bind prothrombin independently of its active site. Tandem mass spectrometry and N-terminal sequencing of purified cotiaractivase identified peptides that were identical to regions of the cysteine-rich and disintegrin-like domains of known snake venom metalloproteinases. Cotiaractivase is a unique low molecular weight snake venom prothrombin activator that likely belongs to the metalloproteinase family of proteins.
Keywords: Snake venom toxin; Cotiaractivase; Prothrombin activator; Bothrops cotiara
C-terminal residues of plant glutamate decarboxylase are required for oligomerization of a high-molecular weight complex and for activation by calcium/calmodulin by Moriyah Zik; Yael Fridmann-Sirkis; Hillel Fromm (pp. 872-876).
Bacterial glutamate decarboxylase (GAD) is a homohexameric enzyme of about 330 kDa. Plant GAD differs from the bacterial enzyme in having a C-terminal extension of 33 amino acids within which resides a calmodulin (CaM)-binding domain. In order to assess the role of the C-terminal extension in the formation of GAD complexes and in activation by Ca2+/CaM, we examined complexes formed with the purified full-length recombinant petunia GAD expressed in E. coli, and with a 9 amino acid C-terminal deletion mutant (GADΔC9). Size exclusion chromatography revealed that the full-length GAD formed complexes of about 580 kDa and 300 kDa in the absence of Ca2+/CaM, whereas in the presence of Ca2+/CaM all complexes shifted to ∼680 kDa. With deletion of 9 amino acids from the C-terminus (KKKKTNRVC500), the ability to bind CaM in the presence of Ca2+, and to purify it by CaM-affinity chromatography was retained, but the formation of GAD complexes larger than 340 kDa and enzyme activation by Ca2+/CaM were completely abolished. Hence, responsiveness to Ca2+/CaM is associated with the formation of protein complexes of 680 kDa, and requires some or all of the nine C-terminal amino acid residues. We suggest that evolution of plant GAD from a bacterial ancestral enzyme involved the formation of higher molecular weight complexes required for activation by Ca2+/CaM.
Keywords: Glutamate decarboxylase (GAD); Calcium (Ca; 2+; ); Calmodulin (CaM); γ-aminobutyrate (GABA)
Alginate-chaperoned facile refolding of Chromobacterium viscosum lipase by Kalyani Mondal; Himadri B. Bohidar; Rajendra P. Roy; Munishwar N. Gupta (pp. 877-886).
Urea denatured lipase from Chromobacterium viscosum lipase could be refolded by addition of alginate with high guluronic acid content. The refolded molecule could be recovered by affinity precipitation. This approach resulted in recovery of 80% (of original activity) as compared to classical dilution method which gave only 21% activity recovery. Dynamic light scattering showed that binding required about 45 min and activity data obtained from affinity precipitation experiments indicated that refolding was almost instantaneous after binding. Circular dichroism (CD) and fluorescence data showed that refolded molecule was identical to the native molecule. It also showed that refolding takes place at the binding stage and not at the precipitation stage. Preliminary studies showed that the refolding strategy worked equally well with lipases from wheat germ and porcine pancreas.
Keywords: Protein refolding; Affinity precipitation; Dynamic light scattering; Circular dichroism; Smart polymers; Lipases
Probing the mechanism of purine nucleoside phosphorylase by steady-state kinetic studies and ligand binding characterization determined by fluorimetric titrations by Beata Wielgus-Kutrowska; Agnieszka Bzowska (pp. 887-902).
Reversible reaction catalyzed by trimeric purine nucleoside phosphorylase (PNP) from Cellulomonas sp. with typical and non-typical substrates, including product inhibition patterns of both reaction directions, and interactions of the enzyme with bisubstrate analogue inhibitors, were investigated by the steady-state kinetic methods and fluorimetric titrations. The ligand chromophores exist most probably as neutral species, and not N(1)-H monoanions, in the complex with PNP, as shown by determination of inhibition constants vs. pH. This supports the mechanism in which hydrogen bond interaction of N(1)-H with Glu204 is crucial in the catalytic process. Stoichiometry of ligand binding, with possible exception of hypoxanthine, is three molecules per enzyme trimer. Kinetic experiments show that in principle the Michaelis–Menten model could not properly describe the reaction. However, this model seems to hold for certain experimental conditions. Data presented here are supported by earlier findings obtained by means of fluorimetric titrations and protective effects of ligands on thermal inactivation of the enzyme. All results are consistent with the following mechanism for trimeric PNPs: (i) random binding of substrates, (ii) potent binding and slow release of some reaction products leading to the circumstances that the chemical step is not the slowest one and that rapid-equilibrium assumptions do not hold, (iii) a dual role of phosphate—a substrate and also a reaction modifier.
Keywords: Abbreviations; Abbreviations for natural purines and nucleosides are used according to IUPAB nomenclature, also when combined with substituents; m; 7; Guo; 7-methylguanosine (m; =; methyl); m; 7; Gua; 7-methylguanine; P; i; orthophosphate; R1P; α-; d; -ribose-1-phosphate; PNP; purine nucleoside phosphorylase; HEPES; (N[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]); hepbs; (N[2-hydroxyethyl]piperazine-N′-[4-butanesulfonic acid]); PME-8-azaDAP; 8-aza-2,6-diamino-(S)-9-[2-(phosphonomethoxy)ethyl]-purine; (S)-HPMP-Gua; 2-amino-6-oxo-9-[3-hydroxy-2-(phosphonomethoxy)propyl]-purine; PME-6-thioGua; 2-amino-9-[2-(phosphonomethoxy)ethyl]-6-sulfanylpurine; K; i; app; apparent inhibition constant; K; m; app; apparent Michaelis constant; K; d; dissociation constant; v; o; initial velocity; c; o; initial substrate concentration; I; inhibitor concentration; U; enzyme activity unitPurine nucleoside phosphorylase; Kinetic mechanism; Catalytic mechanism; Non-hyperbolic kinetics; Random-binding; Trimer
On the edge of the denaturation process: Application of X-ray diffraction to barnase and lysozyme cross-linked crystals with denaturants in molar concentrations by Michèle Salem; Yves Mauguen; Thierry Prangé (pp. 903-912).
Structural data about the early step of protein denaturation were obtained from cross-linked crystals for two small proteins: barnase and lysozyme. Several denaturant agents like urea, bromoethanol or thiourea were used at increasing concentrations up to a limit leading to crystal disruption (≥2 to 6 M). Before the complete destruction of the crystal order started, specific binding sites were observed at the protein surfaces, an indication that the preliminary step of denaturation is the disproportion of intermolecular polar bonds to the benefit of the agent “parasiting� the surface. The analysis of the thermal factors first agree with a stabilization effect at low or moderate concentration of denaturants rapidly followed by a destabilization at specific weak points when the number of sites increase (overflooding effect).
Keywords: Denaturation; Lysozyme; Barnase; Cross-linked crystal; Protein/denaturant interaction; X-ray diffraction; Urea; Thiourea; Bromoethanol
Binding mode of novel 1-substituted quinazoline derivatives to poly(ADP-ribose) polymerase-catalytic domain, revealed by X-ray crystal structure analysis of complexes by Keita Matsumoto; Kazuyuki Kondo; Tomomi Ota; Akira Kawashima; Kunihiro Kitamura; Toshimasa Ishida (pp. 913-919).
In order to clarify the role of the 1-substituent of quinazoline derivatives in their inhibitory activity against poly(ADP-ribose) polymerase (PARP), two novel inhibitors,1 [8-hydroxy-1-(3-morpholinopropyl)-quinazoline-2,4(1H,3H)-dione] and2 [8-hydroxy-1-(3-phenoxypropyl)-quinazoline-2,4(1H,3H)-dione], were synthesized and subjected to X-ray crystal analysis in complex with the PARP C-terminal catalytic domain (PARP-CD), which requires NAD+ coenzyme for biological function. The nicotinamide-mimicking part of the quinazoline skeleton of1 and2 were both located at the nicotinamide subsite of the NAD+-binding pocket in the same manner as previously reported inhibitors: three hydrogen bonds [(Gly-863)NH-O12, (Gly-863)O-HN3 and (Ser-904)Oγ-O12] and stacking interaction between the Tyr-907 phenol and the quinazoline ring. On the other hand, the N-morpholinoprop-3-yl moiety introduced at the 1-position of the quinazoline ring in1 bridged the large gap between the donor site and the acceptor site through a (Met-890)NH-O20(morpholine) hydrogen bond, where the donor and the acceptor sites are classified as the binding sites of NAD+ and the ADP moiety of the poly(ADP-ribose) chain, respectively. In contrast, the N-phenoxyprop-3-yl moiety in2 formed hydrophobic interactions close to the adenosine-binding site of NAD+, unlike the hydrogen bond such as in1. As the inhibitory activities of1 and2 for PARP were much more potent than those of the unsubstituted nicotinamide analogues, these results suggest that the occupation of the proximal region of the ADP phosphate-and adenosine-binding subsite of the donor site or that of the gap between the donor and the acceptor site by the 1-substituent of quinazoline may increase the inhibitory activity considerably. The nearly equal inhibitory activities of1 and2, despite of their different binding modes at the active site, indicate that this 1-substituent is promising in improving the bioavailability of the inhibitor without compromising its inhibitory activity.
Keywords: Abbreviations; PARP; poly(ADP-ribose) polymerase; PARP-CD,PARP-catalytic Domain; 1; 8-hydroxy-1-(3-morpholinopropyl)-quinazoline-2,4(1H,3H)-dione; 2; 8-hydroxy-1-(3-phenoxypropyl)-quinazoline-2,4(1H,3H)-dione; PD128763; 3,4-dihydro-5-methyl-isoquinolinone; 4ANI; 4-amino-1,8-naphthalimide; 3MBA; 3-methoxybenzamide; NU1025; 8-hydroxy-2-methyl-3-hydro-quinazoline-4-one; FR257517; 2-{3-[4-(4-fluorophenyl)-3,6-dihydro-1(2H)-pyridinyl]propyl}-8-methyl-4(3H)-quinazolinone1-substituted quinazoline derivative; inhibitor; PARP; X-ray crystal structure; binding mode
Characterization of the microtubule proteome during post-diapause development of Artemia franciscana by Paul A. O'Connell; Devanand M. Pinto; Ken A. Chisholm; Thomas H. MacRae (pp. 920-928).
The microtubule proteome encompasses tubulin and a diverse group of proteins which associate with tubulin upon microtubule formation. These proteins either determine microtubule organization and function or their activity is influenced by microtubule association. To characterize the microtubule proteome in Artemia franciscana, tubulin assembly was induced with taxol in vitro after 0 and 12 h of post-diapause development. Proteins obtained by extraction of microtubules with 0.5 M NaCl were electrophoresed in two-dimensional gels and analyzed by mass spectrometry. Fifty-five proteins were identified with 10 of these occurring at both developmental stages, and multiple isoforms were observed for some proteins of the Artemia proteome. Their functions include roles in membrane transport, metabolism, chaperoning and protein synthesis, thus reflecting physiological properties of encysted Artemia such as stress resistance and the ability to rapidly initiate post-diapause development. For example, chaperones may protect tubulin during encystment and facilitate folding in metabolically active embryos. Additionally, the interaction of metabolic enzymes with microtubules funnels reaction intermediates, potentially enhancing efficiency within biochemical processes. This study represents the first systematic characterization of a crustacean microtubule proteome. Although it is difficult to be certain that all protein associations documented herein occur in vivo, the results suggest how protein–protein interactions contribute to cytoplasmic organization while implying how Artemia embryos resist stress and remain capable of development once diapause terminates.
Keywords: Microtubule proteome; Two-dimensional gel electrophoresis; Mass spectrometry; Development; Post-diapause cyst; Artemia franciscana
Altered protein profile in chronic myeloid leukemia chronic phase identified by a comparative proteomic study by Luciana Pizzatti; LÃlian Ayres Sá; Jamison Menezes de Souza; Paulo Mascarello Bisch; Eliana Abdelhay (pp. 929-942).
Chronic myeloid leukemia is a hematological disorder in which the Ph chromosome is a marker of the disease, detected virtually in all cases. The chimeric transcripts encode a 210-kDa chimeric protein with altered tyrosine kinase activity, responsible for the disease phenotype. In this work, we tried to identify which are the molecular changes common to chronic phase patients, those that represent the chronic phase molecular phenotype. To address this problem we analyzed through a comparative proteomic approach, several CML bone marrow cells protein profile from patients in chronic phase and healthy bone marrow donors. From these results, we identified 31 differentially expressed proteins. Among these proteins, we pointed out c-Myc binding protein 1, 53BP1, Mdm4, OSBP-related protein 3 and Mortalin as putative candidates to BCR-ABL targets in chronic phase. Moreover, we describe for the first time the cytoplasmic protein map from bone marrow cells that helped in the elucidation of the changes we were looking for.
Keywords: Chronic Myeloid Leukemia; Proteomics; 2D-Electrophoresis; Mass spectrometry; MALDI-TOF; Leukemia
Structural cooperativity in spectrin type repeats motifs of dystrophin by Laleh Saadat; Lena Pittman; Nick Menhart (pp. 943-954).
Dystrophin is a member of the spectrin family of proteins, which are characterized as being predominantly composed the spectrin-type-repeat, a triple α-helical bundle motif present in multiple tandem copies, producing a rod-like shape. Whether or not this motif, which is determined by sequence homology, is correlated with biophysical domains in the intact protein is uncertain. The nature of the domain structure impacts the flexibility and shape of the rod region of this protein, which is a target for modification in several therapeutic approaches aimed at Duchenne Muscular Dystrophy, a common and fatal genetic disease caused by defective dystrophin. We examined three such motifs in dystrophin, expressing them recombinantly both singly and in tandem, and studying their thermodynamic properties by solvent and thermal denaturation. We have found that the degree to which they are independently stable and expressible varies considerably. The fourth motif appears to be largely stable and independent, whereas the third and second motifs interact strongly.
Keywords: Abbreviations; STR; spectrin type repeat; Dx; this refers to the; x; th canonical STR of dystrophin, counting from the amino terminus, with boundaries as set out by Koenig and Kunkel [25].; Dx; big; this refers to an expanded Dx, with 8 or 9 additional flanking amino acids. The sequence of the specific motifs studied in this paper are given in Figure 1; MALS; multi angle light scattering; CD; circular dichroismDystrophin; Spectrin-type-repeat; Stability; Domain; Unfolding
Construction of an active acetohydroxyacid synthase I with a flexible linker connecting the catalytic and the regulatory subunits by Maria Vyazmensky; Stanislav Engel; Olga Kryukov; Dvora Berkovich-Berger; Ludmila Kaplun (pp. 955-960).
Acetohydroxyacid synthase I (AHAS I), one of three isozymes in Escherichia coli catalyzing the first common step in the biosynthesis of branched amino acids, is composed of two kinds of subunits. The large catalytic (B) and small regulatory (N) subunits of the holoenzyme dissociate and associate freely and rapidly and are quite different in size, charge and hydrophobicity, so that high resolution purification methods lead to partial separation of subunits and to heterogeneity. We have prepared several linked AHAS I proteins, in which the large subunit B with a hexahistidine-tag at the N-terminus, was covalently joined by a flexible linker, containing several (X) amino acids, to the small subunit N to form His6-BuXN polypeptides. All linked BuXN polypeptides have similar specific activity, sensitivity to valine and substrate specificity as the wild type holoenzyme. The most successful BuXN linked protein (Bu30N-r) was inserted into and expressed in yeast and its catalytic properties were tested.
Keywords: Linked polypeptide; Valine binding; Yeast; R; -phenylacetyl carbinol
Enzymatic and biochemical properties of a novel human serine dehydratase isoform by Hirofumi Ogawa; Tomoharu Gomi; Mikio Nishizawa; Yumiko Hayakawa; Shunro Endo; Kyoko Hayashi; Hiroshi Ochiai; Fusao Takusagawa; Henry C. Pitot; Hisashi Mori; Hiroaki Sakurai; Keiichi Koizumi; Ikuo Saiki; Hirofumi Oda; Takashi Fujishita; Toshiro Miwa; Muneharu Maruyama; Masashi Kobayashi (pp. 961-971).
A cDNA clone similar to human serine dehydratase (SDH) is deposited in the GenBank/EMBL databases, but its structural and functional bases remain unknown. Despite the occurrence of mRNA, the expected protein level was found to be low in cultured cells. To learn about physicochemical properties of the protein, we expressed the cDNA in Escherichia coli, and compared the expressed protein with that of a hepatic SDH. The purified protein showedl-serine andl-threonine dehydratase activity, demonstrating to be an isoform of SDH. However, their Km and Vmax constants were different in a range of two-order. Removal of Pro128 from the hepatic SDH consisting of 328 residues, which is missing in the corresponding position of the isoform consisting of 329 residues, significantly changed the Michaelis constants and Kd value for pyridoxal 5′-phosphate, whereas addition of a proline residue to the isoform was without effect. These findings suggest the difference in the structures of the active sites of the two enzymes. Another striking feature was that the expressed level of the isoform in E. coli was 7-fold lower than that of the hepatic SDH. Substitution of Val for Leu287 in the isoform dramatically increased the protein level. The high yield of the mutated isoform was also confirmed by the in vitro transcription and translation experiment. The poor expression of the isoform could be explained by the more stable secondary structure of the mRNA than that of the hepatic SDH mRNA. The present findings may provide a clue as to why the protein level in cultured cells is low.
Keywords: Abbreviations; SDH; serine dehydratase; hSDH; hepatic serine dehydratase; cSDH; cancerous serine dehydratase or SDH-like protein 1; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis (and staining); PLP; pyridoxal 5′-phosphate; RT-PCR; reverse-transcription and polymerase-chain reaction; kbp; kilo base-pairs; IPTG; isopropyl-β-D-thiogalctopyranoside; CBB; Coomassie Brilliant Blue (staining); WB; Western blot analysis; Sal; I*; artificial restriction enzyme site similar to; Sal; ISerine dehydratase; Isoform; Recombinant protein; Translational efficiency
Substrate specificity of Aspergillus oryzae family 3 β-glucosidase by James Langston; Neil Sheehy; Feng Xu (pp. 972-978).
Among glycoside hydrolases, β-glucosidase plays a unique role in many physiological and biocatalytical processes that involve the β-linked O-glycosyl bond of various oligomeric saccharides or glycosides. Structurally, the enzyme can be grouped into glycoside hydrolase family 1 and 3. Although the basic (“retaining, double-displacement�) mechanism for the catalysis of family 3 β-glucosidase has been established, in-depth understanding of its structure–function relationship, particularly the substrate specificity that is of great interest for developing the enzyme as a versatile biocatalyst, remains limited. To further probe the active site, we carried out a comparative study on a family 3 β-glucosidase from Aspergillus oryzae with substrates and competitive inhibitors of different structures, in attempt to evaluate the site-specific spatial and chemical interactions between a pyranosyl substrate and the enzyme. Our results showed the enzyme having a strict stereochemical requirement (to accommodate β-d-glucopyranose) for its “−1� active subsite, in contrast to its family 1 counterpart.
Keywords: β-glucosidase; Substrate specificity; Family 3; Aspergillus oryzae
Kinetic study on the irreversible thermal denaturation of Schistosoma japonicum glutathione s-transferase by Indalecio Quesada-Soriano; Federico GarcÃa-Maroto; Luis GarcÃa-Fuentes (pp. 979-984).
The thermal unfolding pathway of the Schistosoma japonicum glutathione S-transferase (Sj26GST) was previously interpreted by applying equilibrium thermodynamics and a reversible two-state model (Kaplan et al., (1997) Protein Science, 6, 399–406), though weak support for this interpretation was provided. In our study, thermal denaturation of Sj26GST has been re-examined by differential scanning calorimetry in the pH range of 6.5–8.5 and in the presence of the substrate and S-hexylglutathione. Calorimetric traces were found to be irreversible and highly scan-rate dependent. Thermogram shapes, as well as their scan-rate dependence, can be globally explained by assuming that thermal denaturation takes place according to one irreversible step described by a first-order kinetic constant that changes with temperature, as given by an Arrhenius equation. On the basis of this model, values for the rate constant as a function of temperature and the activation energy have been determined. Data also indicate that binding of GSH or S-hexylglutathione just exert a very little stabilising effect on the dimeric structure of the molecule.
Keywords: Abbreviations; Sj26GST; glutathione S-transferase from; Schistosoma japonicum; GSH; reduced glutathione; DSC; differential scanning calorimetryDifferential scanning calorimetry; Thermal denaturation; Glutathione S-transferase; Irreversible
Preparation and crystallization of the disulfide-linked HLA-G dimer by Mitsunori Shiroishi; Daisuke Kohda; Katsumi Maenaka (pp. 985-988).
HLA-G is a non-classical MHC class I, which binds to inhibitory receptors, such as Leukocyte Ig-like receptors, to induce a wide range of tolerogenic immunological effects. HLA-G can be expressed as a disulfide-liked dimer both in solution and at the cell surface. However, the three-dimensional structure of the HLA-G dimer is unknown. Here, we report the crystallization of the disulfide-linked dimer form of HLA-G by adding dithiothreitol (DTT), enabling a 3.2-Ã… data set to be collected. We also show that DTT promotes disulfide bond exchange of refolded HLA-G, whose free cysteine was protected, thus facilitating its dimerization. This technique could also be applied for disulfide-mediated dimer/multimer formation of refolded proteins harbouring free cysteines.
Keywords: HLA-G; Maternal–fetal interface; Dithiothreitol (DTT); Dimerization; Crystallization; Refolding
Crystallization and preliminary X-ray crystallographic study of HP1043, a Helicobacter pylori orphan response regulator by Hyang Mi Lee; Eunmi Hong; Byoung-Young Jeon; Dong-Uk Kim; Jung-Sue Byun; Weontae Lee; Hyun-Soo Cho (pp. 989-991).
The HP1043 protein of Helicobacter pylori is related to the two-component system that regulates prokaryotic transcription in response to environmental changes. The HP1043 gene encodes an orphan response regulator (RR). The N-terminal domain of HP1043 (HP1043N) was purified by affinity-column chromatography. The purified protein was crystallized by hanging drop vapor diffusion at 293 K. The crystal belongs to primitive orthorhombic space group P21212 with cell dimensions of a=89.0, b=41.3, and c=31.7 Ã…. Assuming the presence of one molecule per asymmetric unit, the solvent content was estimated to be about 45%. A complete data set was collected at 1.8 Ã… resolution.
Keywords: HP1043; Helicobacter pylori; Two-component system; Orphan response regulator