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1.
The effect of temperature on the kinetics of human plasma butyrylcholinesterase-catalyzed reactions was studied. The Arrhenius plot of o-nitrophenylbutyrate hydrolysis presents a break at 21 degrees C. However, nucleophilic competition data indicate that there is no change in the rate-limiting step of the overall reaction. In addition, the temperature dependence of the bimolecular rate constant of enzyme carbamylation shows a break at 18 degrees C. These results argue for the existence of thermally induced conformational active states of the enzyme tetramer. It is suggested that the effects of this transition on kinetics arise at the acylation step. 相似文献
2.
Salt induced transitions of chromatin core particles studied by tyrosine fluorescence anisotropy. 总被引:8,自引:2,他引:6
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Chromatin core particles containing 146 base pairs of DNA have been found to undergo a single defined transition below 10 mM ionic strength as studied by both sedimentation velocity and tyrosine fluorescence anisotropy. A method is described for the preparation of such core particles from chicken erythrocytes with greater than 50% yield. 相似文献
3.
The low-salt transition of chromatin core particles is reversible if the monovalent cation concentration is kept above 0.2 mM. Exposure of the particles to salt concentrations below this value results in a nonreversible secondary transition. The nonreversible changes are relatively slow with a half-time of about 15 minutes. Once exposed to such low ionic strength, the particles then begin to refold with increasing salt in at least two steps over a much higher ionic strength range than is required for the usual low-salt transition. The refolding is very fast, with a half-time less than a minute. Small differences between particles which had or had not been exposed to very low salt persist even when the particles are returned to near physiological ionic strengths. 相似文献
4.
The binding of a tetrapeptide lysyltryptophylglycyllysine to nucleosome core particles has been investigated using UV absorption and fluorescence spectroscopy. Modifications of the absorption spectra and fluorescence quenching of the tryptophyl residue are consistent with stacking between the indole ring and nucleic acid bases. Therefore DNA interactions with histones do not prevent stacking of the tryptophyl residue with nucleic acid bases in the peptide-core particle complexes. The number of peptide binding sites is reduced to half that of naked DNA. 相似文献
5.
Magnesium binding and conformational change of DNA in chromatin 总被引:1,自引:0,他引:1
The structure of chromatin in the presence of Mg2+ ions was examined by circular dichroism and equilibrium dialysis. Circular dichroism (CD) shows that above 260 nm the intensity of the spectrum of DNA in nucleoproteins decreases as the Mg2+ concentration increases. This change is an intrinsic characteristic of DNA since it is also observed in protein-free DNA and has been attributed to a change in the winding angle of base pairs around the DNA axis. Some structural elements of the DNA in the nucleosome core, therefore, are as movable as those of protein-free DNA. The basic organization of H1-depleted chromatin, 146 base pairs (bp) of DNA wound around core histones and a residual 49 bp in the linker region in the repeating unit, is maintained both in the presence and in the absence of Mg2+ ions, as shown by the fact that the CD spectrum of H1-depleted chromatin has the same type of linear combination between the spectrum of protein-free DNA and that of the nucleosome core in 0.2 mM MgCl2-10 mM triethanolamine (pH 7.8) as it has in 1 mM ethylenediaminetetraacetic acid-10 mM tris(hydroxymethyl) aminomethane (pH 7.8). The ellipticity of chromatin shows a smaller decrease relative to the other nucleoproteins and protein-free DNA upon the addition of Mg2+ ions. Therefore, some structural elements of chromatin are apparently somewhat protected against the conformational change induced by these ions. The spectrum of chromatin becomes almost indistinguishable from that of H1-depleted chromatin in 0.2 mM MgCl2.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
6.
Chromatin core particles near physiological ionic strength undergo a reversible transition induced by changes in pH near neutrality. While sedimentation studies indicate no significant effect on size or shape, changes in tyrosine fluorescence anisotropy and in circular dichroism suggest a somewhat looser structure at high pH. Further support of this suggestion is given by high salt dissociation experiments; at pH 8 core particles begin to show changes at lower salt concentration than at pH 6. The pH transition appears unaffected by the presence of Mg2+ but can be blocked by crosslinking of the histones. A possible relationship is suggested between this transition and increases in intracellular pH which correlate with enhancement in several aspects of cellular activity including DNA replication. 相似文献
7.
Maitra S Saha B Santra CR Mukherjee A Goswami S Chanda PK Karmakar P 《International journal of biological macromolecules》2007,41(1):23-29
Alprazolam (ALP) is a widely prescribed sedative and antidepressant benzodiazepine group of drugs. The wide uses of this drug lead us to investigate its possible interaction with hemoglobin (Hb). Spectrophotometric and spectofluorimetric studies showed strong binding of ALP with Hb. Circular dichroic spectra showed that alpha-helical structure of Hb-subunits has been largely changed. On ALP treatment partial pressure of O(2) is increased in the blood indicating release of O(2) from erythrocytes. Further, the binding of ALP-induced conformational changes in Hb resulting in larger Hb particle size was demonstrated by dynamic light scattering experiment. Thus, the present study unambiguously raises question of danger of random usage of ALP, which binds with and changes the function of Hb. 相似文献
8.
Although the thermal unfolding/aggregation behavior of proteins in solution has been extensively studied, little is known about proteins immobilized on the surface of nanoparticles and other solid-phase materials. In this study we carefully monitor and analyze the thermal denaturation process of three model proteins adsorbed onto aluminum hydroxide as a function of temperature by FT-IR spectroscopy. The results reveal that the proteins immobilized onto aluminum hydroxide retain their native conformation at lower temperatures (<45 °C). Upon thermal denaturation, the structural transition between the native and denatured states is very similar, in terms of disappearance of the major native secondary structural elements, between the proteins adsorbed onto aluminum hydroxide adjuvant and in solution. This result suggests that the thermal stability of proteins is not significantly affected, or marginally affected at most, by the adsorption onto aluminum hydroxide adjuvant, considering a 5 °C temperature interval used for data collection. However, the adsorption rate and crowding of proteins on aluminum hydroxide particles have a profound effect on the aggregation behavior of the proteins, hydrogen bonding strength of intermolecular β-sheet aggregates and conformation of intermediate states. 相似文献
9.
The fluorescence of NADH bound to phosphoglycerate dehydrogenase (3-phosphoglycerate: NAD+ oxidoreductase, EC 1.1.1.95) decreased by 42% between pH 8.5 and 7.0 Serine, an allosteric inhibitor, quenched the fluorescence of enzyme-bound NADH by 29% at pH 8.5, but not at all at pH 7.0. The kinetics of the fluorescence change which occurred when the pH of an enzyme-NADH solution was rapidly shifted from 8.5 to 7.0 was measured using stopped-flow fluorimetry. The kinetics were first order, with a rate constant of 2.83 s-1. This rate constant was similar in magnitude to the rate constants for fluorescence quenching at pH 8.5 by saturating concentrations of serine and glycine, another allosteric inhibitor (Dubrow, R. and Pizer, L.I. (1977) J. Biol. Chem. 252, 1527-1538). These results indicate that the conformation of phosphoglycerate dehydrogenase at pH 7.0 is similar to, but not identical with, the serine-induced conformation at pH 8.5. 相似文献
10.
Conventional kinesin, a dimeric molecular motor, uses ATP-dependent conformational changes to move unidirectionally along a row of tubulin subunits on a microtubule. Two models have been advanced for the major structural change underlying kinesin motility: the first involves an unzippering/zippering of a small peptide (neck linker) from the motor catalytic core and the second proposes an unwinding/rewinding of the adjacent coiled-coil (neck coiled-coil). Here, we have tested these models using disulfide cross-linking of cysteines engineered into recombinant kinesin motors. When the neck linker motion was prevented by cross-linking, kinesin ceased unidirectional movement and only showed brief one-dimensional diffusion along microtubules. Motility fully recovered upon adding reducing agents to reverse the cross-link. When the neck linker motion was partially restrained, single kinesin motors showed biased diffusion towards the microtubule plus end but could not move effectively against a load imposed by an optical trap. Thus, partial movement of the neck linker suffices for directionality but not for normal processivity or force generation. In contrast, preventing neck coiled-coil unwinding by disulfide cross-linking had relatively little effect on motor activity, although the average run length of single kinesin molecules decreased by 30-50%. These studies indicate that conformational changes in the neck linker, not in the neck coiled-coil, drive processive movement by the kinesin motor. 相似文献
11.
McNulty BC Tripathy A Young GB Charlton LM Orans J Pielak GJ 《Protein science : a publication of the Protein Society》2006,15(3):602-608
Natively disordered proteins are a growing class of anomalies to the structure-function paradigm. The natively disordered protein alpha-synuclein is the primary component of Lewy bodies, the cellular hallmark of Parkinson's disease. We noticed a dramatic difference in dilute solution 1H-15N Heteronuclear Single Quantum Coherence (HSQC) spectra of wild-type alpha-synuclein and two disease-related mutants (A30P and A53T), with spectra collected at 35 degrees C showing fewer cross-peaks than spectra acquired at 10 degrees C. Here, we show the change to be the result of a reversible conformational exchange linked to an increase in hydrodynamic radius and secondary structure as the temperature is raised. Combined with analytical ultracentrifugation data showing alpha-synuclein to be monomeric at both temperatures, we conclude that the poor quality of the 1H-15N HSQC spectra obtained at 35 degrees C is due to conformational fluctuations that occur on the proton chemical shift time scale. Using a truncated variant of alpha-synuclein, we show the conformational exchange occurs in the first 100 amino acids of the protein. Our data illustrate a key difference between globular and natively disordered proteins. The properties of globular proteins change little with solution conditions until they denature cooperatively, but the properties of natively disordered proteins can vary dramatically with solution conditions. 相似文献
12.
Paraquat (1,1'-dimethyl-4,4'-bipyridilium dichloride) is a broad-spectrum herbicide that is highly toxic to animals (including man), the major lesion being in the lung. In mammalian cells, paraquat causes deep alterations in the organization of the cytoskeleton, marked decreases in cytoskeletal protein synthesis, and alterations in cytoskeletal protein composition; therefore, the involvement of the cytoskeleton in cell injury by paraquat was suggested. We previously demonstrated that monomeric actin binds paraquat; moreover, prolonged actin exposure to paraquat, in depolymerizing medium, induces the formation of actin aggregates, which are built up by F-actin. In this work we have shown that the addition of paraquat to monomeric actin results in a strong quenching of Trp-79 and Trp-86 fluorescence. Trypsin digestion experiments demonstrated that the sequence 61-69 on actin subdomain 2 undergoes paraquat-dependent conformational changes. These paraquat-induced structural changes render actin unable to completely inhibit DNase I. By using intermolecular cross-linking to characterize oligomeric species formed during paraquat-induced actin assembly, we found that the herbicide causes the formation of actin oligomers characterized by subunit-subunit contacts like those occurring in oligomers induced by polymerizing salts (i.e., between subdomain 1 on one actin subunit and subdomain 4 on the adjacent subunit). Furthermore, the oligomerization of G-actin induced by paraquat is paralleled by ATP hydrolysis. 相似文献
13.
The structure of the nucleosome core particle in solution has been studied by neutron scattering using the full-contrast variation technique, which reduces the experimental spectra to three fundamental scatter functions holding information on shape and structure. Systematic calculations of the fundamental scatter functions expected from proposed core-particle models have been compared with the observed functions and show that the neutron-scattering criteria severely restrict the number of models which can be valid for the structure in solution. The best model for the core particle in solution has a hydrophobic histone core about which 1.7 ± 0.1 turns of DNA are wrapped at a pitch between 3.0 and 3.5 nm. This core contains most of the histone and has an average thickness of 4 nm and diameter 6.4–7.5 nm. While solution scattering is not able to specify uniquely the actual shape of the core to high resolution, all models which are possible for the shape of the core to a resolution justified by the data have been considered. It is clear that cylindrical or wedge shapes compatible with the above dimensions are valid structures. A hole probably penetrates the histone core, but the data do not allow a diameter greater than 1 nm. Available evidence suggests that about a quarter of the total histone is outside the core. 相似文献
14.
We have investigated an inhibition of photophosphorylation which occurs during preillumination of isolated spinach chloroplasts. Preillumination for 4–6 min in the absence of a complete set of components required for ATP synthesis inhibits photophosphorylation to a maximum of 25–40%; no inhibition occurs if all components for phosphorylation are present from the time illumination begins. The inhibition is about 40% recoverable by imposing a dark (“rebound”) period after the preillumination. Photoinhibition is accompanied by an increased leakiness of the thylakoid membrane to protons and is prevented by the presence of FCCP during the preillumination. Several lines of evidence implicate changes in conformation of chloroplast coupling factor (CF1) as the cause of both photoinhibition and dark rebound. Conditions which result in photoinhibition also result in a loss of Mg2+-dependent ATPase activity which can be elicited from chloroplasts. Both photoinhibition and dark rebound are accompanied by changes in the Km of CF1 for both ADP and Pi. Photoinhibition precludes further inhibition of phosphorylation by light plus N-ethylamleimide (NEM) while phosphorylating activity regained by dark rebound is sensitive to subsequent inhibition by light plus NEM. The results are consistent with the conformational coupling hypothesis in indicating that CF1 may be able to store energy in a conformational state which can be released by the reversal of that state. The photoinhibition we observe may represent conformational changes in CF1 which are related to conformational coupling but which lead to photoinhibition under our conditions of preillumination. 相似文献
15.
1H NMR investigation of the conformational states of DNA in nucleosome core particles. 总被引:3,自引:3,他引:0
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In this study 1H NMR has been used to investigate the conformational state of DNA in nucleosome core particles. The nucleosome core particles exhibit partially resolved low field (10-15 ppm) spectra due to imino protons in Watson-Crick base pairs (one resonance per GC or AT base pair). To a first approximation, the spectrum is virtually identical with that of protein-free 140 base pair DNA, and from this observation we draw two important conclusions: (i) Since the low field spectra of DNA are known to be sensitive to conformation, the conformation of DNA in the core particles is essentially the same as that of free DNA (presumably B-form), (ii) since kinks occurring at a frequency at 1 in 10 or 1 in 20 base pairs would result in a core particle spectrum different from that of free DNA we find no NMR evidence supporting either the Crick-Klug or the Sobell models for kinking DNA around the core histones. Linewidth considerations indicate that the rotational correlation time for the core particles is approximately 1.5 X 10(-7) sec, whereas the end-over-end tumbling time of the free 140 base pair DNA is 3 X 10(-7) sec. 相似文献
16.
The cell cycle control phosphatases Cdc25 are dual specificity phosphatases that dephosphorylate both phosphothreonine and phosphotyrosine residues on their substrate proteins. The determination of the apo-protein structure of Cdc25A revealed that this enzyme has a completely different fold compared to all other phosphatases crystallised to date. The conformation of the active site residues does not seem very suitable for catalysis in this unliganded structure. We have studied some structural features of the Cdc25A apo-structure and a modelled Cdc25A-ligand complex by molecular dynamics simulations. The simulations predict a conformational change in the peptide backbone of the complex, which is not observed in the apo-structure. This ligand-induced conformational change yields a structure that is similar to other protein tyrosine phosphatase-ligand complexes that have been crystallised. The change in conformation takes place in the position between a serine and a glutamic acid residue in the phosphate binding loop. We suggest that this type of conformational change is an important molecular switch in the catalytic process. 相似文献
17.
Stability of the primary organization of nucleosome core particles upon some conformational transitions. 总被引:3,自引:3,他引:0
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The sequential arrangement of histones along DNA in nucleosome core particles was determined between 0.5 and 600 mM salt and from 0 to 8 M urea. These concentrations of salt and urea up to 6 M had no significant effect on the linear order of histones along DNA but 8 M urea caused the rearrangement of histones. Conformational changes in cores have been identified within these ranges of conditions by several laboratories 8-21. Also, abrupt structural changes in the cores, apparently their unfolding, were found by gel electrophoresis to occur at urea concentration, between 4 and 5 M. 600 mM salt and 6 M urea were shown to relax the binding of histones to DNA in cores but do not however release histones or some part of their molecules from DNA. It appears therefore that nucleosomal cores can undergo some conformational transitions and unfolding whereas their primary organization remains essentially unaffected. These results are consistent with a model of the core particles in which the histone octamer forms something like a helical "rim" along the superhelical DNA and histone-histone interactions beyond the "rim" are rather weak in comparison with those within the "rim". 相似文献
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20.
Evidence that vector transmission of a plant virus requires conformational change in virus particles
Transmission of Cucumber necrosis virus (CNV) by zoospores of its fungal vector, Olpidium bornovanus, involves specific adsorption of virus particles onto the zoospore plasmalemma prior to infestation of cucumber roots by virus-bound zoospores. Previous work has shown that specific components of both CNV and zoospores are required for successful CNV/zoospore recognition. Here, we show that limited trypsin digestion of CNV following in vitro CNV/zoospore binding assays, results in the production of specific proteolytic digestion products under conditions where native CNV is resistant. The proteolytic digestion pattern of zoospore-bound CNV was found to be similar to that of swollen CNV particles produced in vitro, suggesting that zoospore-bound CNV is in an altered conformational state, perhaps similar to that of swollen CNV. We show that an engineered CNV mutant (Pro73Gly) in which a conserved proline residue (Pro73) in the beta-annulus of the CP arm is changed to glycine is resistant to proteolysis following in vitro zoospore binding assays. Moreover, Pro73Gly particles are transmitted only poorly by O.bornovanus. Together, the results of these studies suggest that CNV undergoes conformational change upon zoospore binding and that the conformational change is important for CNV transmissibility. 相似文献