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1.
Steady-state and time-resolved emission spectroscopy were used to study the interaction of Escherichia coli purine nucleoside phosphorylase (PNP) with its specific inhibitors, viz. formycin B (FB), and formycin A (FA) and its N-methylated analogues, N(1)-methylformycin A (m(1)FA), N(2)-methylformycin A (m(2)FA) and N(6)-methylformycin A (m(6)FA), in the absence and presence of phosphate (P(i)). Complex formation led to marked quenching of enzyme tyrosine intrinsic fluorescence, with concomitant increases in fluorescence of FA and m(6)FA, independently of the presence of P(i). Fluorescence of m(1)FA in the complex increased only in the presence of P(i), while the weak fluorescence of FB appeared unaffected, independently of P(i). Analysis of the emission, excitation and absorption spectra of enzyme-ligand mixtures pointed to fluorescence resonance energy transfer (FRET) from protein tyrosine residue(s) to FA and m(6)FA base moieties, as a major mechanism of protein fluorescence quenching. With the non-inhibitor m(2)FA, fluorescence emission and excitation spectra were purely additive. Effects of enzyme-FA, or enzyme-m(6)FA, interactions on nucleoside excitation and emission spectra revealed shifts in tautomeric equilibria of the bound ligands. With FA, which exists predominantly as the N(1)-H tautomer in solution, the proton N(1)-H is shifted to N(2), independently of the presence of P(i). Complex formation with m(6)FA in the absence of P(i) led to a shift of the amino-imino equilibrium in favor of the imino species, and increased fluorescence at 350 nm; by contrast, in the presence of P(i), the equilibrium was shifted in favor of the amino species, accompanied by higher fluorescence at 430 nm, and a higher affinity for the enzyme, with a dissociation constant K(d)=0.5+/-0.1 microM, two orders of magnitude lower than that for m(6)FA in the absence of P(i) (K(d)=46+/-5 microM). The latter was confirmed by analysis of quenching of enzyme fluorescence according to a modified Stern-Volmer model. Fractional accessibility values (f(a)) varied from 0.31 for m(1)FA to 0.70 for FA, with negative cooperative binding of m(1)FA and FB, and non-cooperative binding of FA and m(6)FA. For all nucleoside ligands, the best model describing binding stoichiometry was one ligand per native enzyme hexamer. Fluorescence decays of PNP, FA and their mixtures were best fitted to a sum of two exponential terms, with average lifetimes () affected by their interactions. Complex formation resulted in a 2-fold increase in of FA, and a 2-fold decrease in of enzyme fluorescence. The amplitude of the long-lifetime component also increased, confirming the shift of the tautomeric equilibrium in favor of the N(2)-H species. The findings have been examined in relation to enzyme-nucleoside binding deduced from structural studies.  相似文献   

2.
We undertook cysteine substitution mutagenesis and fluorophore conjugation at selected residue positions to map sites of ligand binding and changes in solvent exposure of the acetylcholine-binding protein from Lymnaea stagnalis, a nicotinic receptor surrogate. Acrylodan fluorescence emission is highly sensitive to its local environment, and when bound to protein, exhibits changes in both intensity and emission wavelength that are reflected in the degree of solvent exclusion and the effective dielectric constant of the environment of the fluorophore. Hence, cysteine mutants were generated based on the acetylcholine-binding protein crystal structure and predicted ligand binding sites, and fluorescence parameters were assayed on the acrylodan-conjugated proteins. This approach allows one to analyze the environment around the conjugated fluorophore side chain and the changes induced by bound ligand. Introduction of an acrylodan-cysteine conjugate at position 178 yields a large blue shift with alpha-bungarotoxin association, whereas the agonists and alkaloid antagonists induce red shifts reflecting solvent exposure at this position. Such residue-selective changes in fluorescence parameters suggest that certain ligands can induce distinct conformational states of the binding protein, and that mutually exclusive binding results from disparate portals of entry to and orientations of the bound alpha-toxin and smaller acetylcholine congeners at the binding pocket. Labeling at other residue positions around the predicted binding pocket also reveals distinctive spectral changes for alpha-bungarotoxin, agonists, and alkaloid antagonists.  相似文献   

3.
Ferulic acid (FA) is a biologically active compound used as an additive in the food industry, and possesses a wide range of therapeutic effects for treating different health problems. The interaction between FA and bovine xanthine oxidase (XOD) has been investigated by means of fluorescence spectroscopy methods. The numbers of binding sites and the binding constants were estimated at various temperatures and the results indicated the existence of one specific FA binding site of XOD. Detailed information on the interaction between molecules gathered after performing in silico molecular docking indicated the accommodation of the FA molecule in a XOD binding pocket, in close vicinity to the active site residues. The formation of the XOD–FA complex causes the quenching of protein fluorescence. The process followed a static mechanism at lower temperatures, and a dynamic mechanism at higher temperatures. The thermodynamic parameters calculated on the basis of different temperatures revealed that the association between FA and XOD is a spontaneous process driven by enthalpy and dominated by hydrogen bonding and van der Waals interaction. The results of synchronous fluorescence and 3D fluorescence spectra showed that the conformation of protein was altered in the presence of FA. Copyright © 2016 John Wiley & Sons, Ltd.  相似文献   

4.
5.
Ghatak  H.  Mukhopadhyay  S.K.  Jana  T.K.  Sen  B.K.  Sen  S. 《Wetlands Ecology and Management》2004,12(3):145-155
Humic (HA) and fulvic (FA) acids isolated from mangrove sediments of Sundarban, the largest delta on earth in the estuarine phase of the river Ganges, were studied and attempts were made to characterize their binding sites by quenching of Synchronous fluorescence (SyF) bands with Fe (III) and Cu (II). A modified Stern-Volmer relationship applicable for static quenching was applied for the determination of conditional stability constants and the data were compared with those determined by potentiometric titration. In the excited state HA and FA showed different acidity constant compared to the ground state. Values of the conditional stability constant (log Kc) for Fe (III) and Cu (II) indicated that binding sites were bidentate in nature. FA were better chelators than the HA fractions. High energy binding sites of both FA & HA were occupied by Fe(III) and the low energy binding sites, mainly responsible for mobilization and immobilization of metal, were occupied by Cu(II).  相似文献   

6.
Summary The usefulness of the formaldehyde (FA) and glyoxylic acid (GA) methods for the fluorescence histochemical demonstration of dopa thioethers has been tested using protein droplet models. Similar fluorescence intensities were recorded from these compounds after either FA or GA treatment. Cysteinyldopa gave a high fluorescence yield similar to that obtained from dopamine and dopa in the FA reaction, whereas glutationedopa showed a lower, although clearly visible fluorescence. Since the FA method seemed to be the most useful one for demonstration of catechol thioethers, the FA-induced fluorophores of these compounds were further characterized by microspectrofluorometry. The spectral characteristics of the thioether fluorophores (excitation maxima at 420 nm and emission maxima at 480–485 nm) distinguish these substances from dopa and other compounds fluorogenic in the Falck-Hillarp method. Dopa thioethers are proposed to form fluorophores with FA in a manner analogous to that of the primary catecholamines i.e. via low-fluorescent tetrahydroisoquinolines, along two different pathways, to strongly fluorescent 3,4-dihydroisoquinolines and 2-methyl-dihydroisoquinolinium compounds. These dihydroisoquinolines are in a pH-dependent tautomeric equilibrium with their quinoidal forms as reflected by a characteristic spectral shift upon acidification. The results of this study provide the guide-lines for the characterization of fluorogenic compounds in pigment-forming cells.  相似文献   

7.
Quantitative characterization of protein interactions under physiological conditions is vital for systems biology. Fluorescence photobleaching/activation experiments of GFP-tagged proteins are frequently used for this purpose, but robust analysis methods to extract physicochemical parameters from such data are lacking. Here, we implemented a reaction-diffusion model to determine the contributions of protein interaction and diffusion on fluorescence redistribution. The model was validated and applied to five chromatin-interacting proteins probed by photoactivation in living cells. We found that very transient interactions are common for chromatin proteins. Their observed mobility was limited by the amount of free protein available for diffusion but not by the short residence time of the bound proteins. Individual proteins thus locally scan chromatin for binding sites, rather than diffusing globally before rebinding at random nuclear positions. By taking the real cellular geometry and the inhomogeneous distribution of binding sites into account, our model provides a general framework to analyze the mobility of fluorescently tagged factors. Furthermore, it defines the experimental limitations of fluorescence perturbation experiments and highlights the need for complementary methods to measure transient biochemical interactions in living cells.  相似文献   

8.
The environment of the biotin binding site on avidin was investigated by determining the fluorescence enhancement of a series of fluorescent probes that are anilinonaphthalene sulfonic acid derivatives. Of the compounds tested, 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS) exhibited the greatest enhancement under the conditions used (which would reflect both molar fluorescence enhancement and binding affinity) and exhibited more than 95% reversal upon addition of biotin. Thus, 2,6-ANS was chosen for more detailed characterization of the interaction with avidin. Only a single class of binding sites for 2,6-ANS was identified; the mean value for the Kd was 203 +/- 16 microM (X +/- 1 S.D.), and the molar ratio of 2,6-ANS binding sites to biotin binding sites was approx. 1. These results provide evidence that the biotin binding site and the 2,6-ANS binding site are at least partially overlapping, but the possibility that the probe binding site is altered by a conformational change induced by biotin binding cannot be excluded. At excitation = 328 nm and emission = 408 nm, the molar fluorescence of the bound probe was 6.8 +/- 1.0 microM-1 and that of the free probe was 0.061 +/- 0.008 microM-1 giving an enhancement ratio (molar fluorescence of bound probe/molar fluorescence of free probe) of 111 +/- 22. Upon binding, the wavelength of maximum fluorescence decreases. These findings also provide evidence that the fluorescence enhancement associated with the interaction of 2,6-ANS and avidin reflects the environment of the biotin binding site. The Kosower's Z factor, an empirical index of apolarity, was 82.1 for the 2,6-ANS binding site on avidin. This value reflects a degree of apolarity that is similar to apolar environments observed for substrate binding sites on several enzymes; although not the dominant factor, this environment may contribute to the strong binding of biotin.  相似文献   

9.
The denaturation characteristics of inorganic pyrophosphatase from baker's yeast and the interaction with Cu2+ were investigated with fluorimetric methods. The position of the fluorescence emission spectrum with a maximum at 328 nm together with a quantum yield of 0.12 led to the conclusion that most of the tryptophan residues of the protein are buried in nonpolar inner regions of the molecule. The contribution of the tyrosine residues to the fluorescence of pyrophosphatase is only about 7%. Denaturation of the protein with denaturants or changes of the pH value cause a red shift of the fluorescence emission maximum. In the presence of Cu2+ ions a fluorescence quenching is observed. Thereby, a specific binding of one Cu2+ per subunit may be distinguished from further unspecific Cu2+ binding. The Cu2+ binding to the latter sites shows a time dependence according to a slow, reversible exposure of additional binding sites. This time dependent binding characteristics was also verified by following the free Cu2+ concentration with the fluorescent "metal indicator" epsilon-ADP.  相似文献   

10.
Radiationless energy transfer from tyrosine to Tb(III) in Escherichia coli glutamine synthetase and its two mutants (W57L and W158S) has been utilized to assess the tyrosine residue(s) responsible for the observed tyrosine emission and to investigate its spatial relationships to the two metal binding sites of GS. The interference from tryptophan fluorescence was removed by chemical modification of the tryptophan residues by N-bromosuccinimide (NBS). The Tyr-Tb(III) distances measured by using F?rster energy-transfer theory were in good agreement among the three enzymes with average distances of 10.7 and 11.2 A from Tyr to the two metal binding sites. The pKa value for the ionization of tyrosine was determined from fluorescence titration experiments to be approximately 10 for both mutant enzymes. The similarities in pKa values and Tyr-Tb(III) distances observed for all three enzymes lead to the conclusion that the same tyrosine residue(s), is (are) most likely responsible for the Tyr emission. According to the crystal structure distances from tyrosine residues to the two metal binding sites of GS, it is believed that Tyr-179 is the main contributor to the observed Tyr emission. The fact that an intense Tyr emission was observed for W57L GS but not for W158S GS indicates that Trp-57 is much more effective than Trp-158 in quenching the Tyr-179 emission probably through a F?rster-type energy transfer. Furthermore, modification of Trp-57 by NBS causes no significant increase in Tyr-179 emission while replacement of Trp-57 by leucine does. This may indicate that oxidized Trp-57 is also an effective quencher for Tyr-179 emission.  相似文献   

11.
Song XZ  Andreeva IE  Pedersen SE 《Biochemistry》2003,42(14):4197-4207
Fluorescent energy transfer measurements of dansyl-C6-choline binding to the nicotinic acetylcholine receptor (AChR) from Torpedo californica were used to determine binding characteristics of the alpha gamma and alpha delta binding sites. Equilibrium binding measurements show that the alpha gamma site has a lower fluorescence than the alpha delta site; the emission difference is due to differences in the intrinsic fluorescence of the bound fluorophores rather than differences in energy transfer at the two sites. Stopped-flow fluorescence kinetics showed that dissociation of dansyl-C6-choline from the AChR in the desensitized conformation occurs 5-10-fold faster from the alpha gamma site than from the alpha delta site. The dissociation rates are robust for distinct protein preparations, in the presence of noncompetitive antagonists, and over a broad range of ionic strengths. Equilibrium fluorescent binding measurements show that dansyl-C6-choline binds with higher affinity to the alpha delta site (K = 3 nM) than to the alpha gamma site (K = 9 nM) when the AChR is desensitized. Similar affinity differences were observed for acetylcholine itself. The distinct dissociation rates permit the extent of desensitization to be measured at each site during the time course of binding. This sequential mixing method of measuring the desensitized state population at each agonist site can be applied to study the mechanism of AChR activation and subsequent desensitization in detail.  相似文献   

12.
The binding site distribution of concanavalin agglutinin (Con A) and wheat germ agglutinin (WGA) on embryo sacs at various developmental stages of Torenia fournieri L was studied by using a cooled Charge Coupled Device (CCD) and fluorescent Con A and WGA probes. The distribution patterns of Con A and WGA binding sites on embryo sacs changed during the fertilization process. The fluorescent signal indicating Con A binding sites was distributed evenly on the surface of the embryo sac wall before anthesis, was much denser on the micropylar end of the embryo sac wall and looked like a corona on the day of anthesis. After pollination, stronger fluorescence was present on the micropylar end of the embryo sac wall and the filiform apparatus (FA), showing an obvious polar distribution. When the pollen tube entered the embryo sac and reached a synergid, the fluorescence was still concentrated on the micropylar end and FA, and started to appear on the synergid. After fertilization, the polar distribution of the fluorescence gradually disappeared and an even distribution pattern was observed again on the embryo sac wall. These results revealed that the dynamic distribution of Con A binding sites was temporally coupled with the process of fertilization. WGA binding site distribution on the embryo sac was also investigated and showed a simple pattern but also regularly changed during the process of fertilization. The variation of these lectin binding sites during the fertilization process suggests that lectin binding site interactions may play a role in the process.  相似文献   

13.

Background

The molecular details of fatty acid (FA) interactions with albumin are fundamental to understanding transport in the plasma and cellular utilization of these key nutrients and building blocks of membranes.

Scope of review

This review focuses on the development and application of NMR methods to study FA binding to albumin [bovine (BSA) and human (HSA)]. The key strategy was to use 13C enrichment of a specific carbon in the FA as a non-perturbing probe to permit visualization of the small ligand complexed to the very large protein. NMR contributions to illuminating molecular interactions and FA dynamics are summarized from three decades of studies.

Major conclusions

Our early studies detected multiple binding sites that we hypothesized were distinguished because of the unique tertiary structure of the protein in close proximity to the FA labeled carbon in each site. Later crystallographic structures revealed the presence of polar and charged amino acid side chains near the carboxyl carbon of the FA and unique tertiary structures lining all of the FA binding pockets. In collaboration with the crystallography group, several FA sites in the crystalline state were matched with NMR resonances in the solution state. With the newest application of NMR, 2D NMR spectroscopy detected nine binding sites, and three were located in the crystal structure through displacement of drugs with identified sites.

General significance

NMR spectroscopy utilizing the FA as a probe allows characterization of site-specific interactions, molecular motions within binding sites, the order of filling and removal of FA from sites. This article is part of a Special Issue entitled Serum Albumin.  相似文献   

14.
The binding of 8-anilino-1-naphthalenesulfonate (ANS) to ciliary dynein ATPase leads to a marked increase in the dye's fluorescence intensity, accompanied by a blue shift in the observed fluorescence emission maximum. We found that dynein has 37 +/- 3 ANS binding sites and that experimentally applied ANS concentrations failed to alter enzyme activity. The fluorescence properties of the enzyme-dye complex were used to learn more about the binding characteristics of dynein substrates and effectors and to probe for possible conformational changes of the enzyme. The fluorescence of the dynein-ANS complex is increased by a number of substrates, including ATP, GTP, and UTP. The transfer of excitation energy from dynein chromophores to adsorbed ANS was also investigated. Our findings indicate that dynein appears to undergo a localized conformational change in its interaction with ATP. Native dynein was also found to be conformationally different from heat-activated or NEM-modified enzyme as evidenced by the emission and excitation spectra of the various enzyme-ANS complexes.  相似文献   

15.
Engineered glucosamine 6-phosphate deaminase of Escherichia coli with unique reactive cysteines at positions 164 or 206 was created by site-directed mutagenesis to monitor the allosteric transition in solution by the fluorescence emission of the bimane or dansyl-amidoethyl groups attached to the indicated residues. The selection of both positions was due to the differential interaction of these residues between T- and R-conformers at the interface of two trimers that form the hexameric structure; in the T-conformer, residue 164 or 206 presents only intrasubunit contacts, but in the R-conformer, new intersubunit contacts are established. As in the wild-type enzyme, fluorescent-labeled mutants show no modification on the allosteric activation of the K-system, only the kcat was reduced to a value of 72 s(-1) (approximately 50% of wild-type). With these preparations, conformational changes were detected by the fluorescence emission spectra at steady state when the active site or the allosteric site ligands were titrated. Despite the similar changes in the fluorescence spectra that were correlated with the induction of the R-state, differences were observed at the maximal change in the fluorescence spectra and in the relative solvent polarities at the positions labeled. These data suggested structural differences in the conformation of the R-state when it is induced from the active site or from the allosteric site, which is not consistent with the two-state structural model proposed by previous crystallographic studies of this enzyme.  相似文献   

16.
Liu R  Siemiarczuk A  Sharom FJ 《Biochemistry》2000,39(48):14927-14938
P-glycoprotein is a member of the ATP binding cassette family of membrane proteins, and acts as an ATP-driven efflux pump for a diverse group of hydrophobic drugs, natural products, and peptides. The side chains of aromatic amino acids have been proposed to play an important role in recognition and binding of substrates by P-glycoprotein. Steady-state and lifetime fluorescence techniques were used to probe the environment of the 11 tryptophan residues within purified functional P-glycoprotein, and their response to binding of nucleotides and substrates. The emission spectrum of P-glycoprotein indicated that these residues are present in a relatively nonpolar environment, and time-resolved experiments showed the existence of at least two lifetimes. Quenching studies with acrylamide and iodide indicated that those tryptophan residues predominantly contributing to fluorescence emission are buried within the protein structure. Only small differences in Stern-Volmer quenching constants were noted on binding of nucleotides and drugs, arguing against large changes in tryptophan accessibility following substrate binding. P-glycoprotein fluorescence was highly quenched on binding of fluorescent nucleotides, and moderately quenched by ATP, ADP, and AMP-PNP, suggesting that the site for nucleotide binding is located relatively close to tryptophan residues. Drugs, modulators, hydrophobic peptides, and nucleotides quenched the fluorescence of P-glycoprotein in a saturable fashion, allowing estimation of dissociation constants. Many compounds exhibited biphasic quenching, suggesting the existence of multiple drug binding sites. The quenching observed for many substrates was attributable largely to resonance energy transfer, indicating that these compounds may be located close to tryptophan residues within, or adjacent to, the membrane-bound domains. Thus, the regions of P-glycoprotein involved in nucleotide and drug binding appear to be packed together compactly, which would facilitate coupling of ATP hydrolysis to drug transport.  相似文献   

17.
Genetically engineered cytochrome b5 has been used to quantitative binding interactions of this protein with cytochrome P-450cam and sperm whale metmyoglobin by static fluorescence titration. Two cytochrome b5 mutants were constructed by cassette mutagenesis to replace a surface threonine residue with cysteine at two crystallographically defined positions, 65 and 8, located 11 and 21 A, respectively, from the nearest heme edge. The T65C and T8C mutant proteins were labeled with the sulfhydryl selective fluorescent reagent, acrylodan, which provided a spectral probe for monitoring protein-protein association. The fluorescence emission spectra of the acrylodan-labeled T65C mutant exhibited an ionic strength-dependent, blue-shifted fluorescence enhancement upon binding met-myoglobin, cytochrome c, and cytochrome P-450cam, whereas the acrylodan-labeled T8C mutant fluorescence emission remained unchanged during all titrations. Dissociation constants of 1.3, 0.6, and 0.5 microM, pH 7.15, were measured for metmyoglobin, cytochrome P-450cam, and cytochrome c, respectively. A similar averaged binding surface for cytochrome P-450cam and cytochrome c is suggested by their closely related degree of fluorescence enhancement, degree of emission blue shift, and binding free energies. Myoglobin binds less tightly, enhances fluorescence to a greater extent, and exhibits a larger blue shift in acrylodan emission spectra suggesting a different averaged binding orientation relative to the acrylodan probe.  相似文献   

18.
《Ecological Indicators》2008,8(1):100-103
Fluctuating asymmetry (random differences between symmetric structures, FA) is one of the stress indices used recently to assess a subtle effect of environmental degradation on organisms and is expected to increase under stress conditions. In this study, we developed an original technique of measuring FA in seagrass, Halophila ovalis. We analysed five metric and meristic characters on leaves of the seagrass from a polluted and several control locations in a lagoon in Eastern Australia. The seagrass was sampled from three sites at each location. The analyses revealed significant spatial heterogeneity of samples in fluctuating asymmetry with the highest variability was observed among sites. There was no increase in FA of H. ovalis from polluted location. Possible explanations suggest that whether existing concentrations of heavy metals do not cause developmental stress in seagrass or their effect is compensated or even surpassed by effect of uncontrolled factors.  相似文献   

19.
Steady state fluorescence polarization spectra and time-resolved emission decay kinetics have been measured in vitro from malignant and normal rat kidney tissue. The degrees of polarization and emission lifetimes from the cancerous and normal systems are different. The spectroscopic differences are attributed to environmental transformations local to the native flavin and porphyrin fluorophors' binding sites.  相似文献   

20.
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