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Fluctuation domains in myoglobin. Fluorescence quenching studies 总被引:1,自引:0,他引:1
The dynamics of two domains in the myoglobin molecule, close to the heme and inside the protein medium including the surface, are investigated through the study of the fluorescence oxygen quenching of two probes imbedded in the heme pocket: zinc protoporphyrin IX (with a fluorescence lifetime of 2.1 ns) and metal-free protoporphyrin IX (with a fluorescence lifetime of 17.8 ns). 相似文献
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Fluorescence studies on heavy meromyosin-substrate interaction 总被引:15,自引:0,他引:15
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The degree of polarization of the intrinsic fluorescence of purified myosin was estimated. On addition of ATP, polarization of the fluorescence of myosin increased when excited at wavelengths longer than 300 nm. In kinetic studies, coupled with the decay of the increased intensity of fluorescence of myosin, the increased polarization of the fluorescence decreased when the ATP was depleted. The decay of the increased polarization of fluorescence of myosin was specific to MgATP. According to the theory of polarization of the fluorescence of proteins, it is likely that some tryptophan residues of myosin, which are responsible for the increase in the fluorescence intensity and polarization when myosin interacts with substrates, reduce their local freedom of rotation. 相似文献
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The interaction of two commercially available cellulose-based polycations, Polymer JR-400 and Celquat L200, with polyacrylates (PAA-Na) and polyvinylsulphonates (PVS-Na) of various molecular weights was investigated by covalently labelling the polycations with dansyl hydrazine and then studying the fluorescence polarisation of the dansyl group. Celquat L200 was shown to form complexes that were more stoichiometric than the complexes formed by Polymer JR-400 at pH 6·1. This was attributed to the higher charge density and lower average molecular weight of the Celquat L200. At pH 3·5, no complex formation was observed with any of the samples of PAA-Na; PVS-Na samples did form complexes with the polycations and the ones with Polymer JR-400 were more stoichiometric than the complexes formed at pH 6·1.The critical electrolyte concentration (C.E.C.) of each of the complexes was studied using sodium chloride as the electrolyte. The C.E.C. values for Polymer JR-400 complexes were in the order: PAA-Na (230 000)>PVS-Na (15 000)>PAA-Na (90 000)>PVS-Na (4300)>PAA-Na (5000). For complexes of Celquat L200, the order was: PAA-Na (230 000)>PVS-Na (15 000)=PVS-Na (4300)>PAA-Na (90 000)>PAA-Na (5000). The numerical values of the C.E.C. for complexes of Celquat L200 were found to be greater than the values for complexes of Polymer JR-400, thus implying that Celquat L200 binds more strongly to the polyanions. The order of binding indicated that, for a given molecular weight, the complexes formed by the polyvinylsulphonates are stronger than those formed by the polyacrylates. 相似文献
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Computational studies of the interaction of myoglobin and xenon 总被引:2,自引:0,他引:2
R F Tilton U C Singh S J Weiner M L Connolly I D Kuntz P A Kollman N Max D A Case 《Journal of molecular biology》1986,192(2):443-456
Computational studies are used to investigate the energies of xenon binding to myoglobin and to describe pathways through the protein interior for a metmyoglobin-xenon complex. Empirical energy calculations indicate a favorable enthalpic contribution of 0.6 to 4.2 kcal/mol to xenon binding for four experimentally determined xenon sites. These calculated enthalpies help to explain the different xenon occupancies observed experimentally. A fifth site, modeled in place of the iron co-ordinated water molecule in the distal cavity, is also predicted to bind xenon. The largest contribution to the binding energy is from van der Waals' interactions with smaller contributions from polarization and protein strain terms. Ligand trajectory calculations as well as a new geometric algorithm define a connecting network of channel-like pathways through the static protein structure. One or two pathways appear to lead most easily from each major internal cavity to the protein surface. The importance of these channels in protein dynamics and their implications as routes for ligand motion are discussed. 相似文献
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Absorption and fluorescence measurements of DNA-Hoechst 33258 complexes at high molar ratio of DNA phosphate to dye are consistent with the existence of two types of bound species. One type (Type I) predominates at high ionic strength, whereas the other (Type II) occurs at low ionic strength. The fluorescence peak (lambda fmax) depends on the excitation wavelength (lambda ex); lambda fmax shifts toward longer wavelength with increasing lambda ex. Optical properties obtained are summarized in the following: for Type I, lambda amax (absorption) = 352 nm, lambda fmax at lambda ex of 335 nm = 460 nm, tau (fluorescence lifetime) = 2.0-2.5 ns; for Type II, lambda amax = 360 nm, lambda fmax at lambda ex of 335 nm = 470 nm, tau = 4.0-5.0 ns. This behavior is interpreted in terms of solvent-solute relaxation. Type I corresponds to less hydrated bound species, while Type II to more hydrated bound species. 相似文献
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The enhancement of fluorescence intensity of the dansyl group due to the formation of trypsin- or trypsinogen-dansyl-L-arginine complex was measured. Dansyl-L-arginine (L-DA) is a product in the trypsin-catalyzed hydrolysis of dansyl-L-arginine methylester. Trypsinogen was found to have only one binding site for L-DA with the dissociation constant of 6.9 x 10(-3)M, which is identical with the Michaelis constant for the trypsin-catalyzed hydrolysis of dansyl-L-arginine amide (Goto, S. and Hess, G.P., unpublished results). This finding and the results of X-ray diffraction studies (1,2) suggest that this binding site is located in the active site of the enzyme. On the other hand, the active enzyme, trypsin, was found to have at least two binding sites for L-DA. One is located in the active site. The dissociation constant for L-DA bound to this site is 6.7 x 10(-3)M. The other site is probably located in the allosteric site of trypsin. The dissociation constant for L-DA bound to this site is 4.8 x 10(-4)M. 相似文献
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Using fluorescence quenching technique the influence of phospholipids on methemoglobin conformation was investigated. The interaction of methemoglobin with model phospholipid membranes was shown to be followed by changes of protein structure-dynamic organization. 相似文献
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Lifan Shih Youngran ChungRenuka Sriram Thomas Jue 《Biochimica et Biophysica Acta (BBA)/General Subjects》2014
Background
Previous studies have shown that palmitate (PA) can bind specifically and non-specifically to Fe(III) MbCN. The present study has observed PA interaction with physiological states of Fe(II) Mb, and the observations support the hypothesis that Mb may have a potential role in facilitating intracellular fatty acid transport.Methods
1H NMR spectra measurements of the Mb signal during PA titration show signal changes consistent with specific and non-specific binding.Results
Palmitate (PA) interacts differently with physiological states of Mb. Deoxy Mb does not interact specifically or non-specifically with PA, while the carbonmonoxy myoglobin (MbCO) interaction with PA decreases the intensity of selective signals and produces a 0.15 ppm upfield shift of the PA methylene peak. The selective signal change upon PA titration provides a basis to determine an apparent PA binding constant, which serves to create a model comparing the competitive PA binding and facilitated fatty acid transport of Mb and fatty acid binding protein (FABP).Conclusions
Given contrasting PA interaction of ligated vs. unligated Mb, the cellular fatty acid binding protein (FABP) and Mb concentration in the cell, the reported cellular diffusion coefficients, the PA dissociation constants from ligated Mb and FABP, a fatty acid flux model suggests that Mb can compete with FABP transporting cellular fatty acid.General significance
Under oxygenated conditions and continuous energy demand, Mb dependent fatty acid transport could influence the cell's preference for carbohydrate or fatty acid as a fuel source and regulate fatty acid metabolism. 相似文献12.
Arosio D Costantini S Kong Y Vindigni A 《The Journal of biological chemistry》2004,279(41):42826-42835
DNA non-homologous end joining starts with the binding of Ku heterodimers to double strand breaks. In this work, we characterized the thermodynamics of the Ku-DNA interaction by fluorescence anisotropy of the probe-labeled DNA. We determined that the microscopic dissociation constant (kd) for the binding of Ku to a DNA binding site of the proper length (>20 bp) ranges from 22 to 29 nm at 300 mm NaCl. The binding isotherms for DNA duplexes with two or three heterodimers were analyzed with two independent models considering the presence and absence of overlapping binding sites. This analysis demonstrated that there is no or very weak nearest-neighbor cooperativity among the Ku molecules. These models can most likely be applied to study the interaction of Ku with duplexes of any length. Furthermore, our salt dependence studies indicated that electrostatic interactions play a major role in the binding of Ku to DNA and that the kd decreases approximately 60-fold as the salt concentration is lowered from 300 to 200 mm. The slope (Gammasalt) of the plot of log kd versus log[NaCl] is 12.4 +/- 0.1. This value is among the highest reported in the literature for a protein-DNA interaction and suggests that approximately 12 ions are released upon formation of the Ku-DNA complex. In addition, comparison of the slope values measured upon varying the type of cation and anion indicated that approximately nine cations and three anions are released from DNA and Ku, respectively, when the complex is formed. 相似文献
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Nanosecond and steady-state fluorescence spectoscopy were used to probe the environment of the tryptophan residues of Escherichia coli DNA-binding protein. A spectral shift and a change in quantum yield of the protein upon binding to DNA or oligonucleotides indicate that the tryptophan residues are near or at the DNA binding site. The observation of two excited-state lifetimes of the protein indicates that there is heterogeneity in the microenvironments of these tryptophan residues. The "short-lifetime" tryptophan residues are more sensitive to the interaction with DNA than the "long-lifetime" residues. The results of solute-perturbation studies with iodide or acrylamide indicate that there are tryptophan residues near the surface of the protein which are heterogeneous in their accessibility to these quenchers and that they become less accessible after DNA binding. Also, lysine residues of the protein have been shown to be essential to DNA binding by chemical-modification studies. Tyrosine, arginine, and cysteine residues appear not to be involved in this binding process. From studies of the decay of fluorescence anisotropy of the binding protein in the presence and absence of DNA, it has been concluded that (a) the tetrameric binding protein does not dissociate into subuniits upon binding to the oligonucleotide d(pT)16 and (b) the binding protein-fd DNA complex possesses "local flexibility" and, therefore, cannot be described as a continuous, rigid rod. 相似文献
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Genistein (5,7,4′-trihydroxyisoflavone) the common soy beans isoflavone has attracted scientific interest due to its antioxidant, estrogenic, antiangiogenic and aniticancer activities. The aim of the present study was to investigate the interaction of genistein with biological (erythrocyte) and model membranes (dimyristoyl- and dipalmitoylphosphatidylcholine). Using Laurdan and Prodan as fluorescent probes, we demonstrated phase behavior and membrane fluidity changes induced by genistein. ESR spectroscopy revealed alterations caused by genistein in membrane domains structure and mobility of spin probes with free radicals located at different depths of membrane. The method of ESR spectra decomposition and computer simulation of the recorded spectra were used in order to visualize domain coexistence by GHOST condensation method. Fluorescence and ESR spectroscopy experiments performed at different temperatures enabled us to observe the effect of isoflavone on phospholipid bilayers in either gel or liquid crystalline phase. It was concluded that genistein preferentially intercalated into lipid headgroup region, to some extent into polar–apolar interface and only in minimal degree into hydrophobic core of the membrane. According to our best knowledge this is the first study on modification of domain structure of membranes by genistein. 相似文献
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Ubiquinone-10 and ubiquinol-10 were incorporated into dipalmitoylphosphatidylcholine vesicles and their interaction with the phospholipids was monitored by fluorescence measurements of diphenylhexatriene used as a probe. It was found that ubiquinone-10 did not perturb the phospholipid thermotropic pretransition but ubiquinol-10 was able to do so. Although, in ethanolic solution, ubiquinone-10 was a better quencher of diphenylhexatriene than ubiquinol-10, when incorporated into phospholipid multibilayers and at temperatures above Tc, ubiquinone-10 produced a smaller decrease in the intensity of the fluorescence probe than ubiquinol-10. Furthermore, the fluorescence anisotropy of the probe was significantly increased by ubiquinol-10 but not by ubiquinone-10. It was concluded that both forms of coenzyme Q have different localizations in the phospholipid bilayer. 相似文献
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Galmiche A Fueller J Santel A Krohne G Wittig I Doye A Rolando M Flatau G Lemichez E Rapp UR 《The Journal of biological chemistry》2008,283(21):14857-14866
The proteins of the RAF family (A-RAF, B-RAF, and C-RAF) are serine/threonine kinases that play important roles in development, mature cell regulation, and cancer. Although it is widely held that their localization on membranes is an important aspect of their function, there are few data that address this aspect of their mode of action. Here, we report that each member of the RAF family exhibits a specific distribution at the level of cellular membranes and that C-RAF is the only isoform that directly targets mitochondria. We found that the RAF kinases exhibit intrinsic differences in terms of mitochondrial affinity and that C-RAF is the only isoform that binds this organelle efficiently. This affinity is conferred by the C-RAF amino-terminal domain and does not depend on the presence of RAS GTPases on the surface of mitochondria. Finally, we analyzed the consequences of C-RAF activation on mitochondria and observed that this event dramatically changes their morphology and their subcellular distribution. Our observations indicate that: (i) RAF kinases exhibit different localizations at the level of cellular membranes; (ii) C-RAF is the only isoform that directly binds mitochondria; and (iii) through its functional coupling with MEK, C-RAF regulates the shape and the cellular distribution of mitochondria. 相似文献
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The cytochrome P-450K containing monooxygenase system of rat kidney cortex microsomes catalyzes the hydroxylation of various saturated fatty acids of medium chain length to the corresponding ω- and (ω-1)-hydroxy derivatives. The hydroxylation activity, as well as the ratio between the two hydroxylated products, vary with the carbon chain length of the fatty acid. Optimal hydroxylation activity is observed with myristic acid which yields the 13- and 14-hydroxylated products at a ratio of about 1. The ω/(ω-1)-hydroxylation ratio decreases with increasing carbon chain length of the fatty acid. On the other hand, with lauric acid as a substrate the ratio between ω- and (ω-1)-hydroxylation does not change significantly with varying time of incubation or substrate concentration, or incubation in a medium containing D2O or after induction of enhanced hydroxylation activity by starvation of the animals. Furthermore, 12-hydroxylauric acid and capric acid—which is almost exclusively ω-hydroxylated by rat kidney cortex microsomes—inhibit both 11- and 12-hydroxylation of lauric acid to a similar extent whereas 11-hydroxylauric acid does not seem to inhibit either 11- or 12-hydroxylation.C10-C16 fatty acids produce the type I spectral change upon addition to rat kidney cortex microsomes and seem to interact with similar amounts of the cytochrome P-450K present in these particles. In agreement with the metabolic studies, 12-hydroxylauric acid interacts with cytochrome P-450K giving rise to a reverse type I spectral change, whereas 11-hydroxylauric acid does not produce an observable spectral change. Finally, results of binding experiments with a series of derivatives of dodecane suggest that type I binding to cytochrome P-450K requires, besides a proper chain length, the presence of a carbonyl group together with an electron pair on a neighboring atom at the end of the carbon chain. A chain length of 14 carbon atoms seems to be optimal and it is suggested that this chain length may correspond to the distance between a possible binding site and the catalytic site of cytochrome P-450K 相似文献