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Unfolding
Kinds of Unfolding Terms modified by Unfolding Selected AbstractsAnomalous electrophoretic behavior of a very acidic protein: Ribonuclease U2ELECTROPHORESIS, Issue 18 2005Lucía García-Ortega Abstract Ribonuclease U2 is a low-molecular-weight acidic protein with three disulfide bridges. This protein displays an anomalous electrophoretic behavior on standard SDS-PAGE. The electrophoretic mobility of the nonreduced protein roughly corresponds to its molecular mass while the migration of the reduced protein would be in accordance with the expected molecular mass of the protein dimer. This study reveals that the protein does not bind SDS under the SDS-PAGE conditions, its electrophoretic mobility being only determined by its electrostatic charge and hydrodynamic properties. In addition, the nonreduced protein cannot be blotted to a membrane. Unfolding of the protein upon reduction of its disulfide bridges enables electrotransference to membranes due to a restricted diffusion along the electrophoresis gel. [source] Evangelism from a WCC Perspective: a Recollection of an Important Ecumenical Memory, and the Unfolding of a Holistic Vision,INTERNATIONAL REVIEW OF MISSION, Issue 382-383 2007Dietrich Werner [source] The Membrane-Bound Lon Protease from Thermoplasma Displays Unfolding ActivityISRAEL JOURNAL OF CHEMISTRY, Issue 2 2006Henrike Besche The membrane-bound Lon protease from Thermoplasma acidophilum (Ta Lon) was shown to unfold and degrade a fusion of the green fluorescent protein with calmodulin (GFP,CaM). Unfolding and degradation were ATP-dependent reactions and could be inhibited by calcium ions, which are known to stabilize calmodulin. Notably, an inverse fusion of the same proteins, i.e., CaM,GFP, as well as GFP or GFP-SsrA, was neither unfolded nor degraded. Thus, Ta Lon seems to unfold and degrade preferentially protein substrates with an extended unstructured C-terminus. A set of Ta Lon variants mutated in critical residues of the AAA+ domain, were tested for their respective ATPase and GFP,CaM unfolding activity. This analysis revealed that the rate of ATP hydrolysis correlated with the efficiency of the GFP,CaM unfolding activity. In summary, we show here that the membrane-bound Ta Lon protease displays an unfolding activity, which is correlated with the rate of ATP hydrolysis. [source] Urea Unfolding of Opsin in Phospholipid Bicelles,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 2 2009Craig McKibbin Opsin is the unstable apo-protein of the light-activated G protein-coupled receptor rhodopsin. We investigated the stability of bovine opsin, solubilized in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/detergent bicelles, against urea-induced unfolding. A single irreversible protein unfolding transition was observed from changes in intrinsic tryptophan fluorescence and far-UV circular dichroism. This unfolding transition correlated with loss of protein activity. Changes in tertiary structure, as indicated by fluorescence measurements, were concomitant with an approximate 50% reduction in ,-helical content of opsin, indicating that global unfolding had been induced by urea. The urea concentration at the midpoint of unfolding was dependent on the lipid/detergent environment, occurring at approximately 1.2 m urea in DMPC/1,2-dihexanoyl-sn-glycero-3-phosphocholine bicelles, while being significantly stabilized to approximately 3.5 m urea in DMPC/3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate bicelles. These findings demonstrate that interactions with the surrounding lipids and detergent are highly influential in the unfolding of membrane protein structure. The urea/bicelle system offers the possibility for a more detailed understanding of the structural changes that take place upon irreversible unfolding of opsin. [source] Unfolding of the loggerhead sea turtle (Caretta caretta) myoglobin: A 1H-NMR and electronic absorbance studyPROTEIN SCIENCE, Issue 9 2002Daniela Delli Castelli Abstract The effect of urea concentration on the backbone solution structure of the cyanide derivative of ferric Caretta caretta myoglobin (at pH 5.4) is reported. By addition of urea, sequential and long-range nuclear Overhauser effects (NOEs) are gradually lost. By using the residual NOE constraints to build the molecular model, a picture of the unfolding pathway was obtained. When the urea concentration is raised to 2.2 M, helices A and B appear largely disordered; helices C, D, and F loose structural constraints at 3.0 M urea. At urea concentration >6 M, the protein appears to be fully unfolded, including the GH hairpin and helix E stabilizing the prosthetic group. Reversible and cooperative denaturation isotherms obtained by following NOE peaks are considerably different from those obtained by monitoring electronic absorption changes. The reversible and cooperative urea-dependent folding-unfolding process of C. caretta myoglobin follows the minimum three-state mechanism N,X,D, where X represents a disordered globin structure (occurring at ,4 M urea) that still binds the heme. [source] Real-Time Atomistic Description of DNA Unfolding,ANGEWANDTE CHEMIE, Issue 28 2010Alberto Perez Simulationen bis in den Mikrosekundenbereich und die Einführung eines Cosolvens ermöglichen die Charakterisierung der Entfaltungspfade kurzer DNA-Moleküle. Es werden zwei Hauptpfade mit Beteiligung unterschiedlicher Substrate nachgewiesen. Sobald der entfaltete Zustand erreicht ist, nimmt die DNA kompakte Strukturen mit Unterschieden zu einem Zufallsknäuel an (siehe Bild). [source] Protein instability during HIC: Hydrogen exchange labeling analysis and a framework for describing mobile and stationary phase effectsBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2007Yunzhi Xiao Abstract Unfolding of marginally stable proteins is a significant factor in commercial application of hydrophobic interaction chromatography (HIC). In this work, hydrogen-deuterium isotope exchange labeling has been used to monitor protein unfolding on HIC media for different stationary phase hydrophobicities and as a function of ammonium sulfate concentration. Circular dichroism and Raman spectroscopy were also used to characterize the structural perturbations experienced by solution phase protein that had been exposed to media and by protein adsorbed on media. As expected, greater instability is seen on chromatographic media with greater apparent hydrophobicity. However, increased salt concentrations also led to more unfolding, despite the well-known stabilizing effect of ammonium sulfate in solution. A thermodynamic framework is proposed to account for the effects of salt on both adsorption and stability during hydrophobic chromatography. Using appropriate estimates of input quantities, analysis with the framework can explain how salt effects on stability in chromatographic systems may contrast with solution stability. Biotechnol. Bioeng. 2007;96: 80,93. © 2006 Wiley Periodicals, Inc. [source] The Effects of Ligand Exchange and Mobility on the Peroxidase Activity of a Bacterial Cytochrome c upon UnfoldingCHEMBIOCHEM, Issue 4 2005Jonathan A. R. Worrall Dr. Abstract The effect on the heme environment upon unfolding Paracoccus versutus ferricytochrome c-550 and two site-directed variants, K99E and H118Q, has been assessed through a combination of peroxidase activity increase and one-dimensional NMR spectroscopy. At pH 4.5, the data are consistent with a low- to high-spin heme transition, with the K99E mutation resulting in a protein with increased peroxidase activity in the absence of or at low concentrations of denaturant. Furthermore, the mobility of the polypeptide chain at pH 4.5 for the wild-type protein has been monitored in the absence and presence of denaturant through heteronuclear NMR experiments. The results are discussed in terms of local stability differences between bacterial and mitochondrial cytochromes c that are inferred from peroxidase activity assays. At pH 7.0, a mixture of misligated heme states arising from protein-based ligands assigned to lysine and histidine is detected. At low denaturant concentrations, these partially unfolded misligated heme forms inhibit the peroxidase activity. Data from the K99E mutation at pH 7.0 indicate that K99 is not involved in heme misligation, whereas histidine coordination is proven by the data from the H118Q variant. [source] Improving Foldamer Synthesis Through Protecting Group Induced Unfolding of Aromatic Oligoamides.CHEMINFORM, Issue 5 2007Aimin Zhang Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF. [source] Strategies of family farms to strengthen their resilienceENVIRONMENTAL POLICY AND GOVERNANCE, Issue 4 2010Ika Darnhofer Abstract Resilience thinking offers a framework to emphasize dynamics and interdependencies across time, space and domains. It is based on understanding social,ecological systems as complex, and future developments as unpredictable, thus emphasizing adaptive approaches to management. In this paper the four clusters of factors that have been identified as building resilience in large-scale social,ecological systems are applied at the farm level. Suggestions on how these factors could be operationalized at the farm level are derived from workshops held with family farmers in Austria. The results show that farmers understand change as unpredictable and unfolding, have a number of strategies to ensure the flexibility and adaptability of their farm and build extensive networks to diversify information and income sources. However, these strategies, while ensuring adaptability and transformability, compete for scarce resources. The farmers thus face trade-offs between strategies that ensure the adaptive capacity of their farm over the long term and those ensuring profitability over the short term. Copyright © 2010 John Wiley & Sons, Ltd and ERP Environment. [source] Specific cleavage of the DNase-I binding loop dramatically decreases the thermal stability of actinFEBS JOURNAL, Issue 18 2010Anastasia V. Pivovarova Differential scanning calorimetry was used to investigate the thermal unfolding of actin specifically cleaved within the DNaseI-binding loop between residues Met47-Gly48 or Gly42-Val43 by two bacterial proteases, subtilisin or ECP32/grimelysin (ECP), respectively. The results obtained show that both cleavages strongly decreased the thermal stability of monomeric actin with either ATP or ADP as a bound nucleotide. An even more pronounced difference in the thermal stability between the cleaved and intact actin was observed when both actins were polymerized into filaments. Similar to intact F-actin, both cleaved F-actins were significantly stabilized by phalloidin and aluminum fluoride; however, in all cases, the thermal stability of the cleaved F-actins was much lower than that of intact F-actin, and the stability of ECP-cleaved F-actin was lower than that of subtilisin-cleaved F-actin. These results confirm that the DNaseI-binding loop is involved in the stabilization of the actin structure, both in monomers and in the filament subunits, and suggest that the thermal stability of actin depends, at least partially, on the conformation of the nucleotide-binding cleft. Moreover, an additional destabilization of the unstable cleaved actin upon ATP/ADP replacement provides experimental evidence for the highly dynamic actin structure that cannot be simply open or closed, but rather should be considered as being able to adopt multiple conformations. Structured digital abstract ,,MINT-7980274: Actin (uniprotkb:P68135) and Actin (uniprotkb:P68135) bind (MI:0407) by biophysical (MI:0013) [source] Conformational stability and multistate unfolding of poly(A)-specific ribonucleaseFEBS JOURNAL, Issue 10 2009Guang-Jun He Poly(A)-specific ribonuclease (PARN) specifically catalyzes the degradation of the poly(A) tails of single-stranded mRNAs in a highly processive mode. PARN participates in diverse and important intracellular processes by acting as a regulator of mRNA stability and translational efficiency. In this article, the equilibrium unfolding of PARN was studied using both guanidine hydrochloride and urea as chemical denaturants. The unfolding of PARN was characterized as a multistate process, but involving dissimilar equilibrium intermediates when denatured by the two denaturants. A comparison of the spectral characteristics of these intermediates indicated that the conformational changes at low concentrations of the chemical denaturants were more likely to be rearrangements of the tertiary and quaternary structures. In particular, an inactive molten globule-like intermediate was identified to exist as soluble non-native oligomers, and the formation of the oligomers was modulated by electrostatic interactions. An active dimeric intermediate unique to urea-induced unfolding was characterized to have increased regular secondary structures and modified tertiary structures, implying that additional regular structures could be induced by environmental stresses. The dissimilarity in the unfolding pathways induced by guanidine hydrochloride and urea suggest that electrostatic interactions play an important role in PARN stability and regulation. The appearance of multiple intermediates with distinct properties provides the structural basis for the multilevel regulation of PARN by conformational changes. [source] Improving thermostability and catalytic activity of pyranose 2-oxidase from Trametes multicolor by rational and semi-rational designFEBS JOURNAL, Issue 3 2009Oliver Spadiut The fungal homotetrameric flavoprotein pyranose 2-oxidase (P2Ox; EC 1.1.3.10) catalyses the oxidation of various sugars at position C2, while, concomitantly, electrons are transferred to oxygen as well as to alternative electron acceptors (e.g. oxidized ferrocenes). These properties make P2Ox an interesting enzyme for various biotechnological applications. Random mutagenesis has previously been used to identify variant E542K, which shows increased thermostability. In the present study, we selected position Leu537 for saturation mutagenesis, and identified variants L537G and L537W, which are characterized by a higher stability and improved catalytic properties. We report detailed studies on both thermodynamic and kinetic stability, as well as the kinetic properties of the mutational variants E542K, E542R, L537G and L537W, and the respective double mutants (L537G/E542K, L537G/E542R, L537W/E542K and L537W/E542R). The selected substitutions at positions Leu537 and Glu542 increase the melting temperature by approximately 10 and 14 °C, respectively, relative to the wild-type enzyme. Although both wild-type and single mutants showed first-order inactivation kinetics, thermal unfolding and inactivation was more complex for the double mutants, showing two distinct phases, as revealed by microcalorimetry and CD spectroscopy. Structural information on the variants does not provide a definitive answer with respect to the stabilizing effects or the alteration of the unfolding process. Distinct differences, however, are observed for the P2Ox Leu537 variants at the interfaces between the subunits, which results in tighter association. [source] Dynamics, stability and iron-binding activity of frataxin clinical mutantsFEBS JOURNAL, Issue 14 2008Ana R. Correia Friedreich's ataxia results from a deficiency in the mitochondrial protein frataxin, which carries single point mutations in some patients. In the present study, we analysed the consequences of different disease-related mutations in vitro on the stability and dynamics of human frataxin. Two of the mutations, G130V and D122Y, were investigated for the first time. Analysis by CD spectroscopy demonstrated a substantial decrease in the thermodynamic stability of the variants during chemical and thermal unfolding (wild-type > W155R > I154F > D122Y > G130V), which was reversible in all cases. Protein dynamics was studied in detail and revealed that the mutants have distinct propensities towards aggregation. It was observed that the mutants have increased correlation times and different relative ratios between soluble and insoluble/aggregated protein. NMR showed that the clinical mutants retained a compact and relatively rigid globular core despite their decreased stabilities. Limited proteolysis assays coupled with LC-MS allowed the identification of particularly flexible regions in the mutants; interestingly, these regions included those involved in iron-binding. In agreement, the iron metallochaperone activity of the Friedreich's ataxia mutants was affected: some mutants precipitate upon iron binding (I154F and W155R) and others have a lower binding stoichiometry (G130V and D122Y). Our results suggest that, in heterozygous patients, the development of Friedreich's ataxia may result from a combination of reduced efficiency of protein folding and accelerated degradation in vivo, leading to lower than normal concentrations of frataxin. This hypothesis also suggests that, although quite different from other neurodegenerative diseases involving toxic aggregation, Friedreich's ataxia could also be linked to a process of protein misfolding due to specific destabilization of frataxin. [source] Small heat shock protein Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actinFEBS JOURNAL, Issue 22 2007Anastasia V. Pivovarova Previously, we have shown that the small heat shock protein with apparent molecular mass 27 kDa (Hsp27) does not affect the thermal unfolding of F-actin, but effectively prevents aggregation of thermally denatured F-actin [Pivovarova AV, Mikhailova VV, Chernik IS, Chebotareva NA, Levitsky DI & Gusev NB (2005) Biochem Biophys Res Commun331, 1548,1553], and supposed that Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin. In the present work, we applied dynamic light scattering, analytical ultracentrifugation and size exclusion chromatography to examine the properties of complexes formed by denatured actin with a recombinant human Hsp27 mutant (Hsp27,3D) mimicking the naturally occurring phosphorylation of this protein at Ser15, Ser78, and Ser82. Our results show that formation of these complexes occurs upon heating and accompanies the F-actin thermal denaturation. All the methods show that the size of actin,Hsp27-3D complexes decreases with increasing Hsp27-3D concentration in the incubation mixture and that saturation occurs at approximately equimolar concentrations of Hsp27-3D and actin. Under these conditions, the complexes exhibit a hydrodynamic radius of ,,16 nm, a sedimentation coefficient of 17,20 S, and a molecular mass of about 2 MDa. It is supposed that Hsp27-3D binds to denatured actin monomers or short oligomers dissociated from actin filaments upon heating and protects them from aggregation by forming relatively small and highly soluble complexes. This mechanism might explain how small heat shock proteins prevent aggregation of denatured actin and by this means protect the cytoskeleton and the whole cell from damage caused by accumulation of large insoluble aggregates under heat shock conditions. [source] R120G ,B-crystallin promotes the unfolding of reduced ,-lactalbumin and is inherently unstableFEBS JOURNAL, Issue 3 2005Teresa M. Treweek ,-Crystallin is the principal lens protein which, in addition to its structural role, also acts as a molecular chaperone, to prevent aggregation and precipitation of other lens proteins. One of its two subunits, ,B-crystallin, is also expressed in many nonlenticular tissues, and a natural missense mutation, R120G, has been associated with cataract and desmin-related myopathy, a disorder of skeletal muscles [Vicart P, Caron A, Guicheney P, Li Z, Prevost MC, Faure A, Chateau D, Chapon F, Tome F, Dupret JM, Paulin D & Fardeau M (1998) Nat Genet20, 92,95]. In the present study, real-time 1H-NMR spectroscopy showed that the ability of R120G ,B-crystallin to stabilize the partially folded, molten globule state of ,-lactalbumin was significantly reduced in comparison with wild-type ,B-crystallin. The mutant showed enhanced interaction with, and promoted unfolding of, reduced ,-lactalbumin, but showed limited chaperone activity for other target proteins. Using NMR spectroscopy, gel electrophoresis, and MS, we observed that, unlike the wild-type protein, R120G ,B-crystallin is intrinsically unstable in solution, with unfolding of the protein over time leading to aggregation and progressive truncation from the C-terminus. Light scattering, MS, and size-exclusion chromatography data indicated that R120G ,B-crystallin exists as a larger oligomer than wild-type ,B-crystallin, and its size increases with time. It is likely that removal of the positive charge from R120 of ,B-crystallin causes partial unfolding, increased exposure of hydrophobic regions, and enhances its susceptibility to proteolysis, thus reducing its solubility and promoting its aggregation and complexation with other proteins. These characteristics may explain the involvement of R120G ,B-crystallin with human disease states. [source] Probing the unfolding region of ribonuclease A by site-directed mutagenesisFEBS JOURNAL, Issue 20 2004Jens Köditz Ribonuclease A contains two exposed loop regions, around Ala20 and Asn34. Only the loop around Ala20 is sufficiently flexible even under native conditions to allow cleavage by nonspecific proteases. In contrast, the loop around Asn34 (together with the adjacent ,-sheet around Thr45) is the first region of the ribonuclease A molecule that becomes susceptible to thermolysin and trypsin under unfolding conditions. This second region therefore has been suggested to be involved in early steps of unfolding and was designated as the unfolding region of the ribonuclease A molecule. Consequently, modifications in this region should have a great impact on the unfolding and, thus, on the thermodynamic stability. Also, if the Ala20 loop contributes to the stability of the ribonuclease A molecule, rigidification of this flexible region should stabilize the entire protein molecule. We substituted several residues in both regions without any dramatic effects on the native conformation and catalytic activity. As a result of their remarkably differing stability, the variants fell into two groups carrying the mutations: (a) A20P, S21P, A20P/S21P, S21L, or N34D; (b) L35S, L35A, F46Y, K31A/R33S, L35S/F46Y, L35A/F46Y, or K31A/R33S/F46Y. The first group showed a thermodynamic and kinetic stability similar to wild-type ribonuclease A, whereas both stabilities of the variants in the second group were greatly decreased, suggesting that the decrease in ,G can be mainly attributed to an increased unfolding rate. Although rigidification of the Ala20 loop by introduction of proline did not result in stabilization, disturbance of the network of hydrogen bonds and hydrophobic interactions that interlock the proposed unfolding region dramatically destabilized the ribonuclease A molecule. [source] The conformational stability of the Streptomyces coelicolor histidine-phosphocarrier proteinFEBS JOURNAL, Issue 11 2004Characterization of cold denaturation, protein interactions Thermodynamic parameters describing the conformational stability of the histidine-containing phosphocarrier protein from Streptomyces coelicolor, scHPr, have been determined by steady-state fluorescence measurements of isothermal urea-denaturations, differential scanning calorimetry at different guanidinium hydrochloride concentrations and, independently, by far-UV circular dichroism measurements of isothermal urea-denaturations, and thermal denaturations at fixed urea concentrations. The equilibrium unfolding transitions are described adequately by the two-state model and they validate the linear free-energy extrapolation model, over the large temperature range explored, and the urea concentrations used. At moderate urea concentrations (from 2 to 3 m), scHPr undergoes both high- and low-temperature unfolding. The free-energy stability curves have been obtained for the whole temperature range and values of the thermodynamic parameters governing the heat- and cold-denaturation processes have been obtained. Cold-denaturation of the protein is the result of the combination of an unusually high heat capacity change (1.4 ± 0.3 kcal·mol,1·K,1, at 0 m urea, being the average of the fluorescence, circular dichroism and differential scanning calorimetry measurements) and a fairly low enthalpy change upon unfolding at the midpoint temperature of heat-denaturation (59 ± 4 kcal·mol,1, the average of the fluorescence, circular dichroism and differential scanning calorimetry measurements). The changes in enthalpy (m,Hi), entropy (m,Si) and heat capacity (m,Cpi), which occur upon preferential urea binding to the unfolded state vs. the folded state of the protein, have also been determined. The m,Hi and the m,Si are negative at low temperatures, but as the temperature is increased, m,Hi makes a less favourable contribution than m,Si to the change in free energy upon urea binding. The m,Cpi is larger than those observed for other proteins; however, its contribution to the global heat capacity change upon unfolding is small. [source] High thermal and chemical stability of Thermus thermophilus seven-iron ferredoxinFEBS JOURNAL, Issue 23 2003Linear clusters form at high pH on polypeptide unfolding To probe the stability of the seven-iron ferredoxin from Thermus thermophilus (FdTt), we investigated its chemical and thermal denaturation processes in solution. As predicted from the crystal structure, FdTt is extremely resistant to perturbation. The guanidine hydrochloride-induced unfolding transition shows a midpoint at 6.5 m (pH 7, 20 °C), and the thermal midpoint is above boiling, at 114 °C. The stability of FdTt is much lower at acidic pH, suggesting that electrostatic interactions are important for the high stability at higher pH. On FdTt unfolding at alkaline pH, new absorption bands at 520 nm and 610 nm appear transiently, resulting from rearrangement of the cubic clusters into linear three-iron species. A range of iron,sulfur proteins has been found to accommodate these novel clusters in vitro, although no biological function has yet been assigned. [source] The activation of gelsolin by low pHFEBS JOURNAL, Issue 20 2003The calcium latch is sensitive to calcium but not pH Gelsolin is a multidomain and multifunction protein that nucleates the assembly of filaments and severs them. The activation of gelsolin by calcium is a multistep process involving many calcium binding sites that act to unfold the molecule from a tight structure to a more loose form in which three actin-binding sites become exposed. Low pH is also known to activate gelsolin, in the absence of calcium and this too results in an unfolding of the molecule. Less is known how pH-activation occurs but we show that there are significant differences in the mechanisms that lead to activation. Crucially, while it is known that the bonds between G2 and G6 are broken by co-operative occupancy of calcium binding sites in both domains [Lagarrique, E., Maciver, S. K., Fattoum, A., Benyamin, Y. & Roustan, C. (2003) Eur. J. Biochem. 270, 2236,2243.], pH values that activate gelsolin do not result in a weakening of the G2-G6 bonds. We report the existence of pH-dependent conformational changes within G2 and in G4,6 that differ from those induced by calcium, and that low pH overrides the requirement for calcium for actin-binding within G4,6 to a modest extent so that a Kd of 1 µm is measured, compared to 30,40 nm in the presence of calcium. Whereas the pH-dependent conformational change in G2 is possibly different from the change induced by calcium, the changes measured in G4,6 appear to be similar in both calcium and low pH. [source] The relationship between thermal stability and pH optimum studied with wild-type and mutant Trichoderma reesei cellobiohydrolase Cel7AFEBS JOURNAL, Issue 5 2003Harry Boer The major cellulase secreted by the filamentous fungus Trichoderma reesei is cellobiohydrolase Cel7A. Its three-dimensional structure has been solved and various mutant enzymes produced. In order to study the potential use of T. reesei Cel7A in the alkaline pH range, the thermal stability of Cel7A was studied as a function of pH with the wild-type and two mutant enzymes using different spectroscopic methods. Tryptophan fluorescence and CD measurements of the wild-type enzyme show an optimal thermostability between pH 3.5,5.6 (Tm, 62 ± 2 °C), at which the highest enzymatic activity is also observed, and a gradual decrease in the stability at more alkaline pH values. A soluble substrate, cellotetraose, was shown to stabilize the protein fold both at optimal and alkaline pH. In addition, unfolding of the Cel7A enzyme and the release of the substrate seem to coincide at both acidic and alkaline pH, demonstrated by a change in the fluorescence emission maximum. CD measurements were used to show that the five point mutations (E223S/A224H/L225V/T226A/D262G) that together result in a more alkaline pH optimum [Becker, D., Braet, C., Brumer, H., III, Claeyssens, M., Divne, C., Fagerström, R.B., Harris, M., Jones, T.A., Kleywegt, G.J., Koivula, A., et al. (2001) Biochem. J.356, 19,30], destabilize the protein fold both at acidic and alkaline pH when compared with the wild-type enzyme. In addition, an interesting time-dependent fluorescence change, which was not observed by CD, was detected for the pH mutant. Our data show that in order to engineer more alkaline pH cellulases, a combination of mutations should be found, which both shift the pH optimum and at the same time improve the thermal stability at alkaline pH range. [source] A comparison of the urea-induced unfolding of apoflavodoxin and flavodoxin from Desulfovibrio vulgarisFEBS JOURNAL, Issue 1 2002Brian Ó Nuallain The kinetics and thermodynamics of the urea-induced unfolding of flavodoxin and apoflavodoxin from Desulfovibrio vulgaris were investigated by measuring changes in flavin and protein fluorescence. The reaction of urea with flavodoxin is up to 5000 times slower than the reaction with the apoprotein (0.67 s,1 in 3 m urea in 25 mm sodium phosphate at 25 °C), and it results in the dissociation of FMN. The rate of unfolding of apoflavodoxin depends on the urea concentration, while the reaction with the holoprotein is independent of urea. The rates decrease in high salt with the greater effect occurring with apoprotein. The fluorescence changes fit two-state models for unfolding, but they do not exclude the possibility of intermediates. Calculation suggests that 21% and 30% of the amino-acid side chains become exposed to solvent during unfolding of flavodoxin and apoflavodoxin, respectively. The equilibrium unfolding curves move to greater concentrations of urea with increase of ionic strength. This effect is larger with phosphate than with chloride, and with apoflavodoxin than with flavodoxin. In low salt the conformational stability of the holoprotein is greater than that of apoflavodoxin, but in high salt the relative stabilities are reversed. It is calculated that two ions are released during unfolding of the apoprotein. It is concluded that the urea-dependent unfolding of flavodoxin from D. vulgaris occurs because apoprotein in equilibrium with FMN and holoprotein unfolds and shifts the equilibrium so that flavodoxin dissociates. Small changes in flavin fluorescence occur at low concentrations of urea and these may reflect binding of urea to the holoprotein. [source] Pressure-exploration of the 33-kDa protein from the spinach photosystem II particleFEBS JOURNAL, Issue 9 2001Kangcheng Ruan The 33-kDa protein isolated from the spinach photosystem II particle is an ideal model to explore high-pressure protein-unfolding. The protein has a very low free energy as previously reported by chemical unfolding studies, suggesting that it must be easy to modulate its unfolding transition by rather mild pressure. Moreover, the protein molecule consists of only one tryptophan residue (Trp241) and eight tyrosine residues, which can be conveniently used to probe the protein conformation and structural changes under pressure using either fluorescence spectroscopy or fourth derivative UV absorbance spectroscopy. The different experimental methods used in the present study indicate that at 20 °C and pH 6, the 33-kDa protein shows a reversible two-state unfolding transition from atmospheric pressure to about 180 MPa. This value is much lower than those found for the unfolding of most proteins studied so far. The unfolding transition induces a large red shift of the maximum fluorescence emission of 34 nm (from 316 nm to 350 nm). The change in standard free energy (,Go) and in volume (,V) for the transition at pH 6.0 and 20 °C are ,14.6 kJ·mol,1 and ,120 mL·mol,1, respectively, in which the ,Go value is consistent with that obtained by chemical denaturation. We found that pressure-induced protein unfolding is promoted by elevated temperatures, which seem largely attributed to the decrease in the absolute value of ,Go (only a minor variation was observed for the ,V value). However, the promotion of the unfolding by alkaline pH seems mainly related to the increase in ,V without any significant changes in ,Go. It was also found that NaCl significantly protects the protein from pressure-induced unfolding. In the presence of 1 m NaCl, the pressure needed to induce the half-unfold of the protein is shifted to a higher value (shift of 75 MPa) in comparison with that observed without NaCl. Interestingly, in the presence of NaCl, the value of ,V is significantly reduced whilst that of ,Go remains as before. The unfolding-refolding kinetics of the protein has also been studied by pressure-jump, in which it was revealed that both reactions are a two-state transition process with a relatively slow relaxation time of about 102 s. [source] Structural and catalytic properties and homology modelling of the human nucleoside diphosphate kinase C, product of the DRnm23 geneFEBS JOURNAL, Issue 7 2001Muriel Erent The human DRnm23 gene was identified by differential screening of a cDNA library obtained from chronic myeloid leukaemia-blast crisis primary cells. The over-expression of this gene inhibits differentiation and induces the apoptosis of myeloid precursor cell lines. We overproduced in bacteria a truncated form of the encoded protein lacking the first 17 N-terminal amino acids. This truncated protein was called nucleoside diphosphate (NDP) kinase C,. NDP kinase C, had similar kinetic properties to the major human NDP kinases A and B, but was significantly more stable to denaturation by urea and heat. Analysis of denaturation by urea, using size exclusion chromatography, indicated unfolding without the dissociation of subunits, whereas renaturation occurred via a folded monomer. The stability of the protein depended primarily on subunit interactions. Homology modelling of the structure of NDP kinase C,, based on the crystal structure of NDP kinase B, indicated that NDP kinase C, had several additional stabilizing interactions. The overall structure of the two enzymes appears to be identical because NDP kinase C, readily formed mixed hexamers with NDP kinase A. It is possible that mixed hexamers can be observed in vivo. [source] Towards a Semiology of the Periodic Review of UK Regulated UtilitiesFINANCIAL ACCOUNTABILITY & MANAGEMENT, Issue 3 2001Stuart Cooper This note explores the regulatory process of UK privatised utilities through the periodic review of prices. It provides a brief history of the privatisation programme in the UK and the theoretical arguments for the price-cap regulation that has been used. It argues that regulatory process appears to involve a covert dialogue and exchange of information between the regulator and regulated and also a second separate review process that consists of an overt dialogue. Using a semiotic analysis the authors suggest that the unfolding of each of these overt reviews follows a very similar pattern that is constantly being re-enacted. It is concluded that further research is required into the relative importance of the two separate review processes in the setting of the price-cap. [source] Fluctuation of chromatin unfolding associated with variation in the level of gene expressionGENES TO CELLS, Issue 7 2004Noriko Sato We examined whether spontaneous alteration of chromatin structure, if any, correlates with variation in gene expression. Gene activation is associated with changes in chromatin structure at different levels. Large-scale chromatin unfolding is one such change detectable under the light microscope. We established cell clones carrying tandem repeats (more than 50 copies spanning several hundred kb) of the GFP (green fluorescent protein)-ASK reporter genes driven by a tetracycline responsive promoter. These clones constitutively express the transcriptional transactivator. Flow cytometry and live-recording fluorescence microscopy revealed that, although fully activated by a saturating amount of doxycycline, GFP-ASK expression fluctuated in individual cell clones, regardless of the cell cycle stage. The GFP-ASK expression changed from lower to higher levels and vice versa within a few cell cycles. Furthermore, the levels of GFP-ASK expression were correlated with the degrees of chromatin unfolding of the integrated array as detected by FISH (fluorescent in situ hybridization). The chromatin unfolding was not coupled to a mitotic event; around one-third of the daughter-pairs exhibited dissimilar degrees of chromatin unfolding. We concluded that fluctuation of chromatin unfolding was likely to result in variation in gene expression, although the source of the fluctuation of chromatin unfolding remains to be studied. [source] AAA+ superfamily ATPases: common structure,diverse functionGENES TO CELLS, Issue 7 2001Teru Ogura The AAA+ superfamily of ATPases, which contain a homologous ATPase module, are found in all kingdoms of living organisms where they participate in diverse cellular processes including membrane fusion, proteolysis and DNA replication. Recent structural studies have revealed that they usually form ring-shaped oligomers, which are crucial for their ATPase activities and mechanisms of action. These ring-shaped oligomeric complexes are versatile in their mode of action, which collectively seem to involve some form of disruption of molecular or macromolecular structure; unfolding of proteins, disassembly of protein complexes, unwinding of DNA, or alteration of the state of DNA,protein complexes. Thus, the AAA+ proteins represent a novel type of molecular chaperone. Comparative analyses have also revealed significant similarities and differences in structure and molecular mechanism between AAA+ ATPases and other ring-shaped ATPases. [source] Spatial and temporal variability of the phenological seasons in Germany from 1951 to 1996GLOBAL CHANGE BIOLOGY, Issue 6 2001Annette Menzel Abstract Various indications for shifts in plant and animal phenology resulting from climate change have been observed in Europe. This analysis of phenological seasons in Germany of more than four decades (1951,96) has several major advantages: (i) a wide and dense geographical coverage of data from the phenological network of the German Weather Service, (ii) the 16 phenophases analysed cover the whole annual cycle and, moreover, give a direct estimate of the length of the growing season for four deciduous tree species. After intensive data quality checks, two different methods ,,linear trend analyses and comparison of averages of subintervals , were applied in order to determine shifts in phenological seasons in the last 46 years. Results from both methods were similar and reveal a strong seasonal variation. There are clear advances in the key indicators of earliest and early spring (,0.18 to ,0.23 d y,1) and notable advances in the succeeding spring phenophases such as leaf unfolding of deciduous trees (,0.16 to ,0.08 d y,1). However, phenological changes are less strong during autumn (delayed by +,0.03 to +,0.10 d y,1 on average). In general, the growing season has been lengthened by up to ,0.2 d y,1 (mean linear trends) and the mean 1974,96 growing season was up to 5 days longer than in the 1951,73 period. The spatial variability of trends was analysed by statistical means and shown in maps, but these did not reveal any substantial regional differences. Although there is a high spatial variability, trends of phenological phases at single locations are mirrored by subsequent phases, but they are not necessarily identical. Results for changes in the biosphere with such a high resolution with respect to time and space can rarely be obtained by other methods such as analyses of satellite data. [source] Lymphoid microenvironment in the gut for immunoglobulin A and inflammationIMMUNOLOGICAL REVIEWS, Issue 1 2003Robert Chin Summary:, Signaling through lymphotoxin , receptor (LT,R) initiates the unfolding of a host of developmental programs ranging from the organogenesis of lymph nodes and Peyer's patches (PPs) to the coordination of splenic microarchitecture. While investigating an alternative pathway to immunoglobulin A (IgA) production, it was uncovered that LT,R signaling in the lamina propria (LP) stroma orchestrates the coordinated expression of key chemokines and adhesion molecules, creation of a cytokine milieu, and stroma development that facilitates robust IgA production independent of secondary lymphoid structures. Simultaneously, this same infrastructure can be commandeered by autoreactive T cells to organize both the acute destruction of the intestinal mucosa and chronic intestinal inflammation via the ligands for LT,R. The ability to modulate LT,R signaling may alternatively permit the suppression of autoimmune responses and augmentation of gut defenses. [source] Mothers who were severely abused during childhood and their children talk about emotions: Co-construction of narratives in light of maternal traumaINFANT MENTAL HEALTH JOURNAL, Issue 4 2004Nina Koren-Karie The article focuses on detailed examination of the co-construction of emotion dialogues between mothers and their 6-year-old children in light of mothers' experiences of being sexually, physically, and emotionally abused during childhood. We present examples from dialogues between 3 mothers and their children about emotional events experienced by the children and illustrate emotionally mismatched co-construction processes. To better understand these difficulties, the article also provides vignettes from interviews with mothers about children's inner worlds and show how examining the interviews may help explain the unfolding of the dialogues and particularly points of difficulty. The dialogues between the mothers and their children were assessed using the Autobiographical Emotional Events Dialogue procedure (Koren-Karie, Oppenheim, Chaimovich, & Etzion-Carasso, 2000). The maternal interviews were obtained using the Insightfulness Assessment procedure (Oppenheim & Koren-Karie, 2002). The discussion focuses on the significance of mother,child dialogues in shaping children's inner world, and points to the importance of providing intervention for adults who experienced childhood traumas that address not only their own personal issues but also their functioning as parents. [source] | |||||||||||||||||||||||||||||||||||||