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1.
The Aequorea victoria green fluorescent protein (GFP) creates a fluorophore from its component amino acids Ser65, Tyr66, and Gly67 through a remarkable post-translational modification, involving spontaneous peptide backbone cyclization, dehydration, and oxidation reactions. Here we test and extend the understanding of fluorophore biosynthesis by coupling chemical reduction and anaerobic methodologies with kinetic analyses and protein structure determination. Two high-resolution structures of dithionite-treated GFP variants reveal a previously uncharacterized enolate intermediate form of the chromophore that is viable in generating a fluorophore (t1/2 = 39 min-1) upon exposure to air. Isolation of this enolate intermediate will now allow specific probing of the rate-limiting oxidation step for fluorophore biosynthesis in GFP and its red fluorescent protein homologues. Such targeted characterizations may lead to the design of faster maturing proteins with enhanced applications in biotechnology and cell biology. Moreover, our results reveal how the GFP protein environment mimics enzyme systems, by stabilizing an otherwise high energy enolate intermediate to achieve its post-translational modification. 相似文献
2.
Tan L Li Y Drake TJ Moroz L Wang K Li J Munteanu A Chaoyong JY Martinez K Tan W 《The Analyst》2005,130(7):1002-1005
Molecular beacons (MBs) are hairpin-shaped oligonucleotides that contain both fluorophore and quencher moieties. They act like switches and are normally in a closed state, when the fluorophore and the quencher are brought together to turn "off" the fluorescence. When prompted to undergo conformational changes that open the hairpin structure, the fluorophore and the quencher are separated, and fluorescence is turned "on." This Education will outline the principles of MBs and discuss recent bioanalytical applications of these probes for in vitro RNA and DNA monitoring, biosensors and biochips, real-time monitoring of genes and gene expression in living systems, as well as the next generation of MBs for studies on proteins, the MB aptamers. These important applications have shown that MBs hold great potential in genomics and proteomics where real-time molecular recognition with high sensitivity and excellent specificity is critical. 相似文献
3.
The phenomenon of concentration dependent red shift, often observed in synchronous fluorescence spectra (SFS) of monofluorophoric as well as multifluorophoric systems at high chromophore concentrations, is known to have good analytical advantages. This was previously understood in terms of large inner filter effect (IFE) through the introduction of a derived absorption spectral profile that closely corresponds to the SFS profile. Using representative monofluorophoric and multifluorophoric systems, it is now explained how the SF spectral maximum changes with concentration of the fluorophore. For dilute solutions of monofluorophores the maximum is unchanged as expected. It is shown here that the onset of red shift of SFS maximum of both the mono as well as the multifluorophoric systems must occur at the derived absorption spectral parameter value of 0.32 that corresponds to the absorbance value of 0.87. This value is unique irrespective of the nature of the fluorophore under study. For monofluorophoric systems, the wavelength of derived absorption spectral maximum and the wavelength of synchronous fluorescence spectral maximum closely correspond with each other in the entire concentration range. In contrast, for multifluorophoric systems like diesel and aqueous humic acid, large deviations were noted that could be explained as to be due to the presence of non-fluorescing chromophores in the system. This work bridges the entire fluorophore concentration range over which the red shift of SFS maximum sets in; and in the process it establishes the importance of the derived absorption spectral parameter in understanding the phenomenon of concentration dependent red shift of SFS maximum. 相似文献
4.
B Koh G Kim HK Yoon JB Park R Kopelman W Cheng 《Langmuir : the ACS journal of surfaces and colloids》2012,28(32):11676-11686
DNA short oligo, surfactant, peptides, and polymer-assisted dispersion of single-walled carbon nanotube (SWCNTs) in aqueous solution have been intensively studied. It has been suggested that van der Waals interaction, π-π stacking, and hydrophobic interaction are major factors that account for the SWCNTs dispersion. Fluorophore and dye molecules such as Rhodamine B and fluorescein have both hydrophilic and hydrophobic moieties. These molecules also contain π-conjugated systems that can potentially interact with SWCNTs to induce its dispersion. Through a systematic study, here we show that SWCNTs can be dispersed in aqueous solution in the presence of various fluorophore or dye molecules. However, the ability of a fluorophore or dye molecule to disperse SWCNTs is not correlated with the stability of the fluorophore/dye-SWCNT complex, suggesting that the on-rate of fluorophore/dye binding to SWCNTs may dominate the efficiency of this process. We also examined the uptake of fluorophore molecules by mammalian cells when these molecules formed complexes with SWCNTs. The results can have potential applications in the delivery of poor cell-penetrating fluorophore molecules. 相似文献
5.
Efficient Intramolecular Energy Transfer between Two Fluorophores in a Bis‐Branched [3]Rotaxane
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Jian Yao Hong Li Ya‐Nan Xu Prof. Dr. Qiao‐Chun Wang Prof. Dr. Da‐Hui Qu 《化学:亚洲杂志》2014,9(12):3482-3490
A bis‐branched [3]rotaxane, with two [2]rotaxane arms separated by an oligo(para‐phenylenevinylene) (OPV) fluorophore, was designed and investigated. Each [2]rotaxane arm employed a difluoroboradiaza‐s‐indacene (BODIPY) dye‐functionalized dibenzo[24]crown‐8 macrocycle interlocked onto a dibenzylammonium in the rod part. The chemical structure of the [3]rotaxane was confirmed and characterized by 1H and 13C NMR spectroscopy and high‐resolution ESI mass spectrometry. The photophysical properties of [3]rotaxane and its reference systems were investigated through UV/Vis absorption, fluorescence, and time‐resolved fluorescence spectroscopy. An efficient energy‐transfer process in [3]rotaxane occurred from the OPV donor to the BODIPY acceptor because of the large overlap between the absorption spectrum of the BODIPY moiety and the emission spectrum of the OPV fluorophore; this shows the important potential of this system for designing functional molecular systems. 相似文献
6.
Concentrations of fluorophores in histological sections can be determined by measuring the optical density of photomicrographic negatives of the fluorescence emission. This method allows measurements to be made with high resolution and high sensitivity. It is necessary to determine the relationship between fluorophore concentration and emission intensity, the effects of spatial and temporal variations in the intensity of the exciting illumination, and the characteristic curve of each film. A study of these problems with two model systems is described and the procedures necessary to obtain accurate and precise results are reviewed. 相似文献
7.
A minimal core based fluorophore was introduced as a selectively fluorescent "turn on" sensor for Zn(2+) ions in aqueous solution. Addition of Zn(2+) ions to the fluorophore generates a significant emission through a 1:1 ligand-to-metal complex. The fluorescence titration experiment of the minimal core based fluorophore with various metal ions shows that the pyromellitic diimide derivative also has the advantage of a high selectivity to Zn(2+) ions over other metals such as Ni(2+), or Co(2+), Cu(2+), Fe(3+), Fe(2+). More than 8 fold increase in the intensity of fluorescence was observed for the Zn(2+)-bound fluorophore compared to Zn-free fluorophore. Due to its small molecular size, the fluorophore was cell-permeable and successfully applied to the detection of Zn(2+) in living cells. With its relatively high sensitivity to Zn(2+) in living cells, the synthesized new fluorophore will be very useful in the studies on various biological functions of Zn(2+). 相似文献
8.
Several fluorescent signaling systems are built in the format fluorophore-spacer-receptor with ethylenediamine or N,N-dimethylethylenediamine as the receptor, anthracene as the fluorophore, and a methylene group as the spacer. The receptors are derivatized with different electron-withdrawing groups such as 4-nitrobenzene, 4-nitro-2-pyridine, and 2,4-dinitrobenzene, to perturb the photoinduced intramolecular electron transfer (PET) process from the nitrogen lone-pair to the fluorophore. The photophysical properties of these supramolecular systems and their fluorescence responses toward a number of quenching transition metal ions are reported. It is shown that the PET is highly efficient in the absence of a metal ion. With a metal ion input, the fluorescence can be recovered to a different extent depending on the nature of the metal and on the overall architecture of the system as well. Despite the possibility of strong interaction between the fluorophore and the metal ion, significant fluorescence enhancement is observed with quenching of paramagnetic transition metal ions. The complex stability data show that the stability constants for the metal ions showing fluorescence enhancement are of the order of 10(4) M(-1). This study shows that structurally simple fluorescent signaling systems for quenching transition metal ions can be built by maximizing the PET. It is also shown here that simple structural modification can make these systems highly specific for particular transition metal ions for potential applications in several contemporary areas of research. 相似文献
9.
Fluorescence correlation spectroscopy (FCS) is being applied increasingly to study diffusion and interactions of fluorescently labeled macromolecules in complex biological systems. Fluctuations in detected fluorescence, deltaF(t), are expressed as time-correlation functions, G(tau), and photon-count histograms, P(k;DeltaT). Here, we developed a generalized simulation approach to compute G(tau) and P(k;DeltaT) for complex systems with arbitrary geometry, photophysics, diffusion, and macromolecular interactions. G(tau) and P(k;DeltaT) were computed from deltaF(t) generated by a Brownian dynamics simulation of single-molecule trajectories followed by a Monte Carlo simulation of fluorophore excitation and detection statistics. Simulations were validated by comparing analytical and simulated G(tau) and P(k;DeltaT) for diffusion of noninteracting fluorophores in a three-dimensional Gaussian excitation and detection volume. Inclusion of photobleaching and triplet-state relaxation produced significant changes in G(tau) and P(k;DeltaT). Simulations of macromolecular interactions and complex diffusion were done, including transient fluorophore binding to an immobile matrix, cross-correlation analysis of interacting fluorophores, and anomalous sub- and superdiffusion. The computational method developed here is generally applicable for simulating FCS measurements on systems complicated by fluorophore interactions or molecular crowding, and experimental protocols for which G(tau) and P(k;DeltaT) cannot be computed analytically. 相似文献
10.
Wang L Jin J Chen X Fan HH Li BK Cheah KW Ding N Ju S Wong WT Li C 《Organic & biomolecular chemistry》2012,10(28):5366-5370
Optical imaging provides an indispensable way to locate tumors in their early stages with high sensitivity and signal to background ratio. A heptamethine cyanine based fluorophore that emits both single photon near-infrared fluorescence and two-photon deep red fluorescence under physiological conditions was developed. Linear and nonlinear photophysical properties of this fluorophore were investigated and it demonstrated the capability to label lysosomes in cancer cells. The advantages of this fluorophore, including tolerable cytotoxicity, high fluorescence quantum yield, and the ability to emit both near-infrared single photon fluorescence and deep red two photon fluorescence in aqueous solution, give it potential to be used in intra-operatively optical image-guided tumor excision followed by two-photon fluorescence microscopy biopsy analysis after a single administration. 相似文献
11.
Chunlong Sun Peng Wang Lushen Li Gaoxin Zhou Xi Zong Bing Hu Rui Zhang Jin Cai Junqing Chen Min Ji 《Applied biochemistry and biotechnology》2014,172(2):1036-1044
A new near-neutral pH near-infrared (NIR) fluorescent probe utilizing a fluorophore–receptor molecular framework that can modulate the fluorescence emission intensity through a fast photoinduced electron transfer process was developed. Our strategy was to choose tricarbocyanine (Cy), a NIR fluorescent dye with high extinction coefficients, as a fluorophore, and N-methylpiperazine (MP) as a receptor. The pH titration indicated that MP-Cy can monitor the minor physiological pH fluctuations with a pKa of ~7.10 near physiological pH, which is valuable for intracellular pH researches. The probe responds linearly and rapidly to minor pH fluctuations within the range of 3.05–7.10 and exhibits strong dependence on pH changes. As expected, the real-time imaging of cellular pH and the detection of pH in situ was achieved successfully in living HepG2 cells by this probe. It is shown that the probe effectively avoids the influence of autofluorescence and native cellular species in biological systems and meanwhile exhibits high sensitivity, good photostability, and excellent cell membrane permeability. 相似文献
12.
基于DPA识别基团的锌离子荧光传感器 总被引:1,自引:0,他引:1
锌离子在生物体系中扮演着重要角色,其分析和检测在疾病诊断和医疗检测等方面具有重要价值。用于 Zn2+ 检测的荧光传感器具有检测方便、灵敏度高等优点,引起了广泛关注。典型的荧光传感器通常是由识别基团和作为报告单元的荧光团通过间隔基团或直接连接而组成的。识别基团是荧光传感器的作用核心,在高选择性识别过程中起着至关重要的作用。自 1996 年第一次接在荧光素上以来,DPA (N,N-二(2-吡啶甲基)胺, di-2-picolylamine) 基团在锌离子传感器的设计中得到了广泛应用。本文综述了近年来文献中报道的基于 DPA 识别基团的锌离子传感器,介绍了锌离子荧光传感器的合成方法与识别原理,最后简单介绍了锌离子传感器中其它几种常见的识别基团。 相似文献
13.
Vanessa C. Lussini Dr. James P. Blinco Dr. Kathryn E. Fairfull‐Smith Prof. Steven E. Bottle 《Chemistry (Weinheim an der Bergstrasse, Germany)》2015,21(50):18258-18268
Novel profluorescent mono‐ and bis‐isoindoline nitroxides linked to napthalimide and perylene diimide structural cores are described. These nitroxide‐fluorophore probes display strongly suppressed fluorescence in comparison to their corresponding non‐radical diamagnetic methoxyamine derivatives. The perylene‐based probe possessing two isoindoline systems tethered through ethynyl linkages was shown to be the most photostable in solution, demonstrating significantly enhanced longevity over the 9,10‐bis(phenylethynyl)anthracene fluorophore used in previous profluorescent nitroxide probes. 相似文献
14.
Vives G Giansante C Bofinger R Raffy G Del Guerzo A Kauffmann B Batat P Jonusauskas G McClenaghan ND 《Chemical communications (Cambridge, England)》2011,47(37):10425-10427
Selective nucleophilic substitution on a perfluorophenyl unit comprising a BODIPY fluorophore using an alkanethiol or a primary amine offers a quantitative fluorophore labelling strategy, while retaining high photostability and emission quantum yields approaching unity. 相似文献
15.
This study addresses magnetic field effects in exciplex forming donor-acceptor systems. For moderately exergonic systems, the exciplex and the locally excited fluorophore emission are found to be magneto-sensitive. A previously introduced model attributing this finding to excited state reversibility is confirmed. Systems characterised by a free energy of charge separation up to approximately -0.35 eV are found to exhibit a magnetic field effect on the fluorophore. A simple three-state model of the exciplex is introduced, which uses the reaction distance and the asymmetric electron transfer reaction coordinate as pertinent variables. Comparing the experimental emission band shapes with those predicted by the model, a semi-quantitative picture of the formation of the magnetic field effect is developed based on energy hypersurfaces. The model can also be applied to estimate the indirect contribution of the exchange interaction, even if the perturbative approach fails. The energetic parameters that are essential for the formation of large magnetic field effects on the exciplex are discussed. 相似文献
16.
The 1,3-dipolar cycloaddition reaction of bipyridinium ylides with the electron deficient propynamido-β-cyclodextrin was studied. This reaction resulted in the regiospecific formation of a new class of fluorescent β-cyclodextrins. The new fluorophore systems were characterized spectroscopically by their absorption and emission maxima and their quantum yields. 相似文献
17.
自旋标记荧光探针表征生物活性分子的自由基损伤 总被引:2,自引:1,他引:1
生命过程中产生的经基自由基(˙OH)已引起广泛关注.目前,对于˙OH的研究主要集中在直接对˙OH进行定量表征[1~3]和问接检测˙OH诱导损伤生物大分了的损伤产物.˙OH诱导损伤生物大分了,能够产生大量的碳中心自由基[4,5]. 相似文献
18.
Akika Futamura Asuka Uemura Takeshi Imoto Dr. Yusuke Kitamura Dr. Hirotaka Matsuura Dr. Chun‐Xia Wang Toshiki Ichihashi Dr. Yusuke Sato Prof. Dr. Norio Teramae Prof. Dr. Seiichi Nishizawa Prof. Dr. Toshihiro Ihara 《Chemistry (Weinheim an der Bergstrasse, Germany)》2013,19(32):10526-10535
We propose a binary fluorimetric method for DNA and RNA analysis by the combined use of two probes rationally designed to work cooperatively. One probe is an oligonucleotide (ODN) conjugate bearing a β‐cyclodextrin (β‐CyD). The other probe is a small reporter ligand, which comprises linked molecules of a nucleobase‐specific heterocycle and an environment‐sensitive fluorophore. The heterocycle of the reporter ligand recognizes a single nucleobase displayed in a gap on the target labeled with the conjugate and, at the same time, the fluorophore moiety forms a luminous inclusion complex with nearby β‐CyD. Three reporter ligands, MNDS (naphthyridine–dansyl linked ligand), MNDB (naphthyridine–DBD), and DPDB (pyridine–DBD), were used for DNA and RNA probing with 3′‐end or 5′‐end modified β‐CyD – ODN conjugates. For the DNA target, the β‐CyD tethered to the 3′‐end of the ODN facing into the gap interacted with the fluorophore sticking out into the major groove of the gap site ( MNDS and DPDB ). Meanwhile the β‐CyD on the 5′‐end of the ODN interacted with the fluorophore in the minor groove ( MNDB and DPDB ). The results obtained by this study could be a guideline for the design of binary DNA/RNA probe systems based on controlling the proximity of functional molecules. 相似文献
19.
Masaki Takahashi Ayato YamamotoToshiyasu Inuzuka Tetsuya SengokuHidemi Yoda 《Tetrahedron》2011,67(49):9484-9490
Lipophilic bisanthracene-based fluorophore and its derivatives were synthesized by the Suzuki-Miyaura cross-coupling reaction of 9-anthrylboronic acid with a substituted dibromobenzene. In addition to desirable fluorescent properties, these molecular systems were demonstrated to serve as versatile building blocks toward the synthesis of two types of new light-harvesting dendrimers due to their chemical stability. 相似文献
20.
Chatterjee A Suzuki TM Takahashi Y Pacheco Tanaka DA 《Chemistry (Weinheim an der Bergstrasse, Germany)》2003,9(16):3920-3929
Anthracenes bearing aliphatic or aromatic amino substituents, which behave as molecular sensors, have shown their potential to act as photon-induced electron-transfer (PET) systems. In this PET, the fluorophore moieties are responsible for electron release during protonation and deprotonation. The principle of hard and soft acids and bases (HSAB) deals with both intra- and intermolecular electron migration. It is possible to calculate the localized properties in terms of Fukui functions in the realm of density functional theory (DFT) and thus calculate and establish a numerical matchmaking procedure that will generate an a priori rule for choosing the fluorophore in terms of its activity. We calculated the localized properties for neutral, anionic, and cationic systems to trace the course of the efficiency. A qualitative scale is proposed in terms of the feasibility of intramolecular hydrogen bonding. To investigate the effect of the environment of the nitrogen atom on protonation going from mono- to diprotonated systems, we calculated the partial density of states and compared the activity sequence with reactivity indices. The results show that location of the nitrogen atom in an aromatic ring does not influence the PET, but for aliphatic chains it plays a role. Furthermore, the protonation/deprotonation scenario has been explained. The results show that the reactivity indices can be used as a suitable property for scaling the activity of fluorophore molecules for the PET process. 相似文献