共查询到20条相似文献,搜索用时 31 毫秒
1.
Photophysical properties of hydroxyphenyl benzazoles and their applications as fluorescent probes to study local environment in DNA,protein and lipid
下载免费PDF全文
![点击此处可从《Luminescence》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Fluorescence techniques have drawn increasing attention because they provide crucial information about molecular interactions in protein–ligand systems beyond that obtained by other methods. The advantage of fluorescence spectroscopy stems from the fact that the majority of molecules in biological systems do not exhibit fluorescence, making fluorescent probes useful with high sensitivity. Also, the fluorescence emission is highly sensitive to the local environment, providing a valuable tool to investigate the nature of binding sites in macromolecules. In this review, we discuss some of the important applications of a class of molecules that have been used as fluorescent probes in a variety of studies. Hydroxyphenyl benzazoles (HBXs) show distinct spectroscopic features that make them suitable probes for the study of certain biological mechanisms in DNA, protein and lipid. In particular, the complex photophysics of 2‐(2′‐hydroxyphenyl)benzoxazole (HBO) and the distinguished fluorescence signatures of its different tautomeric forms make this molecule a useful probe in several applications. Among these are probing the DNA local environment, study of the flexibility and specificity of protein‐binding sites, and detecting the heterogeneity and ionization ability of the head groups of different lipidic phases. The spectroscopy of HBX molecules and some of their chemically modified structures is also reviewed. Copyright © 2016 John Wiley & Sons, Ltd. 相似文献
2.
We show a new application of fluorescence resonance energy transfer (FRET) in two stages to detect specific sequences of nucleic acids. In the first stage, two fluorescently tagged oligonucleotides hybridize with a complementary target molecule to produce FRET. The sequences of the oligonucleotides and spectral properties of fluorophores are chosen to provide a basis for an efficient energy transfer. In the next step, the specificity of hybridization is tested by competition of labeled probes with an excess of unlabeled oligonucleotides of the same sequence. The resulting emission spectra, one obtained in the excess of unlabeled donor probe and the other produced in the excess of unlabeled acceptor probe, are compared with the spectrum from the first stage to look for differences in the emission pattern of the fluorescent labels. We show that it is possible to detect the existence of specific hybrids composed of the two probes and complementary target molecule even in very unfavorable conditions, such as the presence of unhybridized probes in the final reaction mixture, secondary nonacceptor quenching of donor probe fluorescence, and strong background emission of acceptor produced by its direct excitation with a donor excitation light. 相似文献
3.
Joachimsthal E.L. Ivanov V. Tay S.T.L. Tay J.H. 《World journal of microbiology & biotechnology》2003,19(5):527-533
The use of fluorescence in situ hybridization (FISH) in conjunction with flow cytometry is a popular method of analysing environmental microbial populations. However, false-positive results can be produced if the specificity of oligonucleotide probe binding is not considered. An aim of this research was to evaluate the specificity of labelled oligonucleotide probe binding in FISH by flow cytometry. An excess of unlabelled probe was used to competitively inhibit the specific binding of labelled probe. Comparisons were made between the mean cell fluorescence and the number of fluorescently stained cells in a pure culture of Escherichia coli ATCC 53323. Specific binding of species-specific probes for the detection of E. coli was in the range 47–70% of total binding. A eukaryote probe and a nonsense probe, used as negative controls, had no specific binding with cells of E. coli. The significance of the results obtained is that the enumeration of specifically probe-bound microbial cells by FISH and flow cytometry must be made by an application of labelled and unlabelled probes to distinguish specifically stained cells. This is also a more practical method for the analysis of environmental samples compared to washing of excess non-specifically bound probe, due to the reduction of cell loss from the analysis. 相似文献
4.
Recent findings indicate that ion-chelator probes with tetracarboxylate structure bind proteins. It was suggested that these fluorescent probes are valuable tools to gain information on protein structure through the energy transfer from tryptophans to the bound probe. Here, the binding of the fluorescent probe Mag-Indo-1 to bovine serum albumin (BSA) was investigated. Mag-Indo-1 was reported previously to serve as a probe for magnesium cations (Kd = 2.8 x 10(-4) M for zero ionic strength) which can also interact with calcium cations (Kd = 7.5 x 10(-7) M). Probe complexation with protein results in a shift of the emission fluorescence spectrum of the probe from 480 to 457 nm. We used emission fluorescence techniques to monitor this interaction. Computational resolution of the complex fluorescence spectra and a new software to test the theoretical model were developed in our laboratory. This enabled us to calculate the number of interacting sites and the dissociation constants. The fluorescent probe Mag-Indo-1 binds at a singular site with high affinity (Kd = 1.8 x 10(-7) M) to bovine serum albumin (BSA). Since proteins are known to bind several compounds unspecifically, we have studied the influence of EDTA as a competitor of the probe. Our findings suggest that the BSA binding site is identical for both Mag-Indo-1 and EDTA. We found that EDTA binds the protein with Kd = 0.4 x 10(-3) M. We studied the influence of calcium and found that Mag-Indo-1 does not bind the calcium free Apo-protein anymore. 相似文献
5.
Andrew Nickerson Tao Huang Li-Jung Lin Xioalin Nan 《Journal of visualized experiments : JoVE》2015,(106)
Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 °C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples. 相似文献
6.
In recent years, confocal laser scanning microscopy has been developed into a non-invasive tool to probe intra-particle profiles
of protein in chromatographic adsorbents. A necessary prerequisite when using this technique lies in the labeling of proteins
with fluorescent probes. The quality of the obtained results is thus strongly dependent on the probes used, its sensitivity
on experimental parameters and the change of protein characteristics upon binding. In this review, the fundamental issues
when using fluorescent probes are described, before giving a critical evaluation on published literature in the field of confocal
laser scanning microscopy for the analysis of chromatographic principles. 相似文献
7.
We present here the physicochemical and biochemical properties of NBD-DFO, the 7-nitrobenz-2-oxa-1,3-diazole (NBD) derivative of the siderophore, desferrioxamine B (DFO) (Lytton et al., Mol. Pharmacol. 40, 584, 1991). Modification of DFO at its terminal amine renders it more lipophilic, imparts to it fluorescent properties, and is conservative of the high-affinity iron(III) binding capacity. NBD-DFO partitions readily from aqueous solution into n-octanol (Pcoeff = 5) and displays solvent-induced shifts in absorption and fluorescence spectra. The relative quantum yield of the probe's fluorescence increases over a 10-fold range with decreasing dielectric constant of the solvent. Fluorescence is quenched upon binding of iron(III) to the probe. We demonstrate here the application of NBD-DFO for the specific detection and monitoring of iron (III) in solutions and iron(III) mobilization from cells. Interactions between fluorescent siderophore and the ferriproteins ferritin and transferrin were monitored under physiological conditions. Iron removal from ferritin was evident by the demonstrable quenching of NBD-DFO fluorescence by scavenged iron(III). Quantitation of iron sequestered from cells by NBD-DFO or from other siderophore-iron(III) complexes was accomplished by dissociation of NBD-DFO-Fe complex by acidification and addition of excess ethylenediamin-etetraacetic acid. The sensitivity of the method and the iron specificity indicate its potential for monitoring chelatable iron under conditions of iron-mediated cell damage, iron overload, and diseases of iron imbalance such as malaria. 相似文献
8.
It is shown that conformational changes of receptor proteins brought about by binding of a ligand induce changes in the lipid environment of the receptor that can be monitored by fluorescent lipid probes. On this basis a new approach to studies of ligand-receptor binding is proposed. Using the interaction of the ricin B-chain with Burkitt lymphoma cells as an example and fluorescent labelled sphingomyelin as a probe, the ligand-induced changes of fluorescence anisotropy were shown to be concentration-dependent and to permit determination of the binding constant and the number of receptor-binding sites. The method was found to be specific and highly sensitive, allowing detection of the action of one RB molecule per cell. Scatchard analysis of the binding of 125I-RB demonstrated the presence on the cell surface of two binding sites with Kd approximately 10(-10) and approximately 10(-8) M, respectively. Only the high-affinity sites were detected by the fluorescence technique. Saturation of these sites resulted in maximum inhibition of protein synthesis. 相似文献
9.
Plesniak L Horiuchi Y Sem D Meinenger D Stiles L Shaffer J Jennings PA Adams JA 《Biochemistry》2002,41(47):13876-13882
EnvZ is a histidine protein kinase important for osmoregulation in bacteria. While structural data are available for this enzyme, the nucleotide binding pocket is not well characterized. The ATP binding domain (EnvZB) was expressed, and its ability to bind nucleotide derivatives was assessed using equilbrium and stopped-flow fluorescence spectroscopy. The fluorescence emission of the trinitrophenyl derivatives, TNP-ATP and TNP-ADP, increase upon binding to EnvZB. The fluorescence enhancements were quantitatively abolished in the presence of excess ADP, indicating that the fluorescent probes occupy the nucleotide binding pocket. Both TNP-ATP and TNP-ADP bind to EnvZB with high affinity (K(d) = 2-3 microM). The TNP moiety attached to the ribose ring does not impede access of the fluorescent nucleotide into the binding pocket. The association rate constant for TNP-ADP is 7 microM(-1) s(-1), a value consistent with those for natural nucleotides and the eucaryotic protein kinases. Using competition experiments, it was found that ATP and ADP bind 30- and 150-fold more poorly, respectively, than the corresponding TNP-derivatized forms. Surprisingly, the physiological metal Mg(2+) is not required for ADP binding and only enhances ATP affinity by 3-fold. Although portions of the nucleotide pocket are disordered, the recombinant enzyme is highly stable, unfolding only at temperatures in excess of 70 degrees C. The unusually high affinity of the TNP derivatives compared to the natural nucleotides suggests that hydrophobic substitutions on the ribose ring enforce an altered binding mode that may be exploited for drug design strategies. 相似文献
10.
Minor groove binder-conjugated DNA probes for quantitative DNA detection by hybridization-triggered fluorescence 总被引:1,自引:0,他引:1
Here we describe the properties of a novel class of oligonucleotide probes capable of sensitive hybridization-triggered fluorescence. These fluorogenic probes, known commercially as MGB Eclipse probes, are characterized by having a conjugated minor groove binder (MGB) ligand at the 5'-end and a fluorophore at the 3'-end. Additionally, they have an efficient quencher moiety at the 5'-end that is useful with a wide variety of fluorescent dyes. Fluorescence of the single-stranded MGB Eclipse probe is efficiently quenched by the interaction of the terminal dye and quencher groups when not hybridized. Upon hybridization to a complementary target, the MGB molecule folds into duplex and hyper-stabilizes it, allowing the use of shorter, more specific probe sequences. The 5'-MGB-quencher group also prevents nuclease digestion by Taq DNA polymerase during PCR. Because of the hybridization-triggered fluorescence and the excellent specificity imparted by the MGB, these 5'-MGB Eclipse probes have great versatility for real-time PCR applications. The high sensitivity and specificity are illustrated using single nucleotide polymorphism detection, viral load determination, and gene expression analysis. 相似文献
11.
Periplasmic binding proteins from E. coli undergo large conformational changes upon binding their respective ligands. By attaching a fluorescent probe at rationally selected unique sites on the protein, these conformational changes in the protein can be monitored by measuring the changes in fluorescence intensity of the probe which allow the development of reagentless sensing systems for their corresponding ligands. In this work, we evaluated several sites on bacterial periplasmic sulfate-binding protein (SBP) for attachment of a fluorescent probe and rationally designed a reagentless sensing system for sulfate. Eight different mutants of SBP were prepared by employing the polymerase chain reaction (PCR) to introduce a unique cysteine residue at a specific location on the protein. The sites Gly55, Ser90, Ser129, Ala140, Leu145, Ser171, Val181, and Gly186 were chosen for mutagenesis by studying the three-dimensional X-ray crystal structure of SBP. An environment-sensitive fluorescent probe (MDCC) was then attached site-specifically to the protein through the sulfhydryl group of the unique cysteine residue introduced. Each fluorescent probe-conjugated SBP mutant was characterized in terms of its fluorescence properties and Ser171 was determined to be the best site for the attachment of the fluorescent probe that would allow for the development of a reagentless sensing system for sulfate. Three different environment-sensitive fluorescent probes (1,5-IAEDANS, MDCC, and acylodan) were studied with the SBP171 mutant protein. A calibration curve for sulfate was constructed using the labeled protein and relating the change in the fluorescence intensity with the amount of sulfate present in the sample. The detection limit for sulfate was found to be in the submicromolar range using this system. The selectivity of the sensing system was demonstrated by evaluating its response to other anions. A fast and selective sensing system with detection limits for sulfate in the submicromolar range was developed. 相似文献
12.
Peptide substrates were double labeled with pyrenes to prepare fluorescent probes for highly sensitive detection of protease activity and evaluation of protease inhibitors using pyrene monomer/excimer signals. Two proximate pyrene moieties formed excited state dimers in the probes, and these pyrene excimer formations were dissociated by tryptic digestion. The specificity constant of the optimum bispyrene peptide probe was 2.7 times higher than that of the conventional peptide-4-methylcoumarin amide. Moreover, our probe had high sensitivity with an estimated detection limit for trypsin of 4.11?pM. The half maximal inhibitory concentration and dissociation constant of the Bowman–Birk inhibitor were successfully estimated. 相似文献
13.
Mathejczyk JE Pauli J Dullin C Napp J Tietze LF Kessler H Resch-Genger U Alves F 《Molecular imaging》2011,10(6):469-480
Labeling of RGD peptides with near-infrared fluorophores yields optical probes for noninvasive imaging of tumors overexpressing ανβ3 integrins. An important prerequisite for optimum detection sensitivity in vivo is strongly absorbing and highly emissive probes with a known fluorescence lifetime. The RGD-Cy5.5 optical probe was derived by coupling Cy5.5 to a cyclic arginine-glycine-aspartic acid-d-phenylalanine-lysine (RGDfK) peptide via an aminohexanoic acid spacer. Spectroscopic properties of the probe were studied in different matrices in comparison to Cy5.5. For in vivo imaging, human glioblastoma cells were subcutaneously implanted into nude mice, and in vivo fluorescence intensity and lifetime were measured. The fluorescence quantum yield and lifetime of Cy5.5 were found to be barely affected on RGD conjugation but dramatically changed in the presence of proteins. By time domain fluorescence imaging, we demonstrated specific binding of RGD-Cy5.5 to glioblastoma xenografts in nude mice. Discrimination of unspecific fluorescence by lifetime-gated analysis further enhanced the detection sensitivity of RGD-Cy5.5-derived signals. We characterized RGD-Cy5.5 as a strongly emissive and stable probe adequate for selective targeting of ανβ3 integrins. The specificity and thus the overall detection sensitivity in vivo were optimized with lifetime gating, based on the previous determination of the probes fluorescence lifetime under application-relevant conditions. 相似文献
14.
Detection of nitrifying bacteria in activated sludge by fluorescent in situ hybridization and fluorescence spectrometry 总被引:2,自引:0,他引:2
16S rRNA-targeted oligonucleotide probes for eubacteria (EUB338), ammonium-oxidizing bacteria (Nsm156) and nitrite-oxidizing bacteria (Nb1000) were used for the rapid detection of nitrifying bacteria in the activated sludge of a pilot nitrifying reactor by whole-cell, fluorescent in situ hybridization (FISH). Emission scanning and synchronous scanning fluorescence spectrometry were used to measure the hybridization. The binding of the probes at a temperature significantly lower than the melting temperature of the hybrids was conventionally considered as non-specific. Total binding of the probes at a temperature significantly higher than the melting temperature of the hybrids was conventionally considered as the sum of non-specific and specific binding (hybridization). Non-specific binding of the oligonucleotide probes with a biomass of activated sludge was 37% of the total binding of the EUB338 probe, 54% of the total binding of the Nsm156 probe, and 69% of the total binding of the Nb1000 probe. The ratio of the specific binding of the Nsm156 and Nb1000 probes was 2.3:1. The ratio of the numbers of ammonium-oxidizing bacteria to nitrite-oxidizing bacteria, determined by microbiological methods, was 2.4:1. Measuring fluorescent in situ hybridization by fluorescence spectrometry appears to be a practical tool for monitoring the microbial communities that contain nitrifying bacteria. However, a method that accounts for the non-specific binding of the probes more easily and reliably should be developed for practical application. 相似文献
15.
Thompson M 《Bioconjugate chemistry》2006,17(2):507-513
The design of a dsDNA-sensitive fluorescent bioconjugate capable of targeting a specific DNA sequence with high efficiency is described. The bioconjugate has the molecular recognition features of the polypeptide from a DNA-binding protein and the dsDNA-dependent fluorescence of an intercalating dye. The DNA sequence selectivity of the probe was characterized, as were the changes in photophysical properties of the dye upon covalent linkage to the peptide to assess whether such bioconjugates could function as molecular probes of gene sequences. The oxazole yellow-peptide bioconjugate exhibits DNA recognition and binding affinity comparable to the native Hin recombinase protein. Examination of photophysical effects to dye conjugation indicates a negligible affect on the fluorescence quantum yield. Fluorescence studies indicate this molecular probe is useful to determine the presence of a given DNA target sequence and gives negligible fluorescence in the absence of a given target site. Using the synthetic route described here, bioconjugates could be designed using different combinations of DNA recognition polypeptides and cyanine dyes to generate an array of sequence specific and wavelength specific probes. 相似文献
16.
Bogoeva VP Radeva MA Atanasova LY Stoitsova SR Boteva RN 《Biochimica et biophysica acta》2004,1698(2):213-218
Wheat germ agglutinin (WGA) from embryos of the monocotyledonous plant Triticum vulgaris (Graminaceae) is a carbohydrate binding protein characterized by high specificity to N-acetyl-d-glucosamine and N-acetyl-d-neuraminic acid. In this study we show that parallel to its carbohydrate binding activities, WGA binds with several orders of magnitude higher affinity adenine, adenine-related cytokinins: kinetin, zeatin and isopentenyl-adenine as well as abscisic and gibberellic acids (K(d) 0.43-0.65 microM). Its interactions with these ligands cause conformational rearrangements in the protein molecules and significant enhancement of the protein tryptophan fluorescence (up to 60%) allowing characterization of the protein-hormone complexes. Dimeric WGA molecules possess two different classes of binding sites for the fluorescent hydrophobic probe 2-(p-toluidinyl) naphthalene sulfonic acid (TNS) as suggested by the sigmoid shape of the fluorescence titration curve and the value of the Hill coefficient (n(H) 1.6+/-0.3). The plant hormones displace part of the bound TNS probe and share the higher affinity TNS binding sites. These results characterize WGA as a hormone-binding protein. 相似文献
17.
Here we describe protocols for preparing and using fluorescent probes that respond to conformational changes by altered Foerster resonance energy transfer (FRET) efficiencies upon phosphorylation or, in principle, other posttranslational modifications (PTMs). The sensor protein, a truncated version of pleckstrin, is sandwiched between short-wavelength-excitation green fluorescent protein (GFP2) and yellow fluorescent protein (EYFP). As a result of complex conformational changes of the protein upon phosphorylation, the introduction of a second PTM consensus sequence bestows sensitivity to a second modification and yields a dual-parameter probe. The first phase of the protocol lays out the cloning strategy for single- and dual-parameter FRET sensors, including the construction of a versatile platform into which different consensus sequences may be inserted to create diverse probes. Protocols for fluorescence microscopy of the probes in living cells and image processing are also described. Probe preparation takes 7 d; microscopy and image processing take 2 h. 相似文献
18.
Fluorescently labeled collagen binding proteins allow specific visualization of collagen in tissues and live cell culture 总被引:5,自引:0,他引:5
Krahn KN Bouten CV van Tuijl S van Zandvoort MA Merkx M 《Analytical biochemistry》2006,350(2):177-185
Visualization of the formation and orientation of collagen fibers in tissue engineering experiments is crucial for understanding the factors that determine the mechanical properties of tissues. In this study, collagen-specific fluorescent probes were developed using a new approach that takes advantage of the inherent specificity of collagen binding protein domains present in bacterial adhesion proteins (CNA35) and integrins (GST-alpha1I). Both collagen binding domains were obtained as fusion proteins from an Escherichia coli expression system and fluorescently labeled using either amine-reactive succinimide (CNA35) or cysteine-reactive maleimide (GST-alpha1I) dyes. Solid-phase binding assays showed that both protein-based probes are much more specific than dichlorotriazinyl aminofluorescein (DTAF), a fluorescent dye that is currently used to track collagen formation in tissue engineering experiments. The CNA35 probe showed a higher affinity for human collagen type I than did the GST-alpha1I probe (apparent K(d) values of 0.5 and 50 microM, respectively) and showed very little cross-reactivity with noncollagenous extracellular matrix proteins. The CNA35 probe was also superior to both GST-alpha1I and DTAF in visualizing the formation of collagen fibers around live human venous saphena cells. Immunohistological experiments on rat tissue showed colocalization of the CNA35 probe with collagen type I and type III antibodies. The fluorescent probes described here have important advantages over existing methods for visualization of collagen, in particular for monitoring the formation of collagen in live tissue cultures over prolonged time periods. 相似文献
19.
Juan-Juan Chen Jing Jing Hao Chang Yueguang Rong Yang Hai Juan Tang Jun-Long Zhang Pingyong Xu 《Autophagy》2013,9(6):894-904
Autophagy is a complex, multi-step and biologically important pathway mediated by autophagosomes and autolysosomes. Accurately dissecting and detecting different stages of autophagy is important to elucidate its molecular mechanism and thereby facilitate the discovery of pharmaceutical molecules. We herein reported a small-molecule synthetic probe, Zn-G4, which is only fluorescent upon starvation- or chemical agent-induced autophagy within the autolysosome or possible the late endosome/lysosome networks. The probe can be detected by one-photon microscopy, which gives a high signal-to-noise ratio readout of autophagic activity. The pH gradient-independent fluorescence can be detected both in live and prestained fixed cells. Moreover, the fluorescent recording can be used to quantify autophagic activity at a single point without transfection or false positive signals due to protein aggregation. Furthermore, autophagy-induced fluorescence in autolysosomes can also be detected by two-photon microscopy, suggesting potential applications in deep tissue and in vivo. In conclusion, we have developed a sensitive and specific autolysosomal probe that can be used for monitoring autophagy during later stages along with quantitative assays together with widely used early markers or microtubule-associated protein 1 light chain 3 (LC3)-based probes. 相似文献
20.
Tadao Takada Chie Tanaka Mitsunobu Nakamura Kazushige Yamana 《Bioorganic & medicinal chemistry letters》2010,20(3):994-996
The DNA base stack provides unique features for the efficient long-range charge transfer. For the purpose of investigating excess electron transfer process through DNA, we developed a new method for fluorescence analysis of excess electron transfer based on reductive cleavage of a disulfide bond and a thiol-specific fluorescent probe. Excess electron transfer was detected by monitoring the fluorescence of emissive pyrene monomer generated by the reaction of pyrene maleimides with the cleaved disulfide bond (thiols). Mechanism of reductive cleavage of disulfides through excess electron transfer and subsequent reaction with the fluorescent probes were discussed. This facile and sensitive detection by fluorescence method can be applied for mechanistic study of excess electron transfer. 相似文献