共查询到20条相似文献,搜索用时 0 毫秒
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Arjun Sharma Aranyak Sarkar Dr. Dibakar Goswami Dr. Arunasis Bhattacharyya Dr. Jörg Enderlein Dr. Manoj Kumbhakar 《Chemphyschem》2019,20(16):2093-2102
Fluorescence correlation spectroscopy (FCS) has been extensively used to measure equilibrium binding constants (K) or association and dissociation rates in many reversible chemical reactions across chemistry and biology. For the majority of investigated reactions, the binding constant was on the order of ∼100 M−1, with dissociation constants faster or equal to 103 s−1, which ensured that enough association/dissociation events occur during the typical diffusion-determined transition time of molecules through the FCS detection volume. However, complexation reactions involving metal ions and chelating ligands exhibit equilibrium constants exceeding 104 M−1. In the present paper, we explore the applicability of FCS for measuring reaction rates of such complexation reactions, and apply it to binding of iron, europium and uranyl ions to a fluorescent chelating ligand, calcein. For this purpose, we exploit the fact that the ligand fluorescence becomes strongly quenched after binding a metal ion, which results in strong intensity fluctuations that lead to a partial correlation decay in FCS. We also present measurements for the strongly radioactive ions of 241Am3+, where the extreme sensitivity of FCS allows us to work with sample concentrations and volumes that exhibit close to negligible radioactivity levels. A general discussion of the applicability of FCS to the investigation of metal-ligand binding reactions concludes our paper. 相似文献
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Sina Berndl Hans‐Achim Wagenknecht Prof. 《Angewandte Chemie (International ed. in English)》2009,48(13):2418-2421
A fluorescent chameleon : A single thiazole orange (TO) dye, when used as an artificial DNA base shows the typical green emission, whereas the interstrand TO dimer exhibits an orange excimer‐type emission inside duplex DNA (see picture).
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Photoswitching of cation complexation with A monoaza-crown dithienylethene photochrome 总被引:1,自引:0,他引:1
Malval JP Gosse I Morand JP Lapouyade R 《Journal of the American Chemical Society》2002,124(6):904-905
A photochromic dithienylethene, bearing a phenyl azacrown as an ionophore and a formyl group as an electron-accepting substituent, changes its binding ability for Ca2+ by a factor higher than 103 by photoirradiation. This new photoionochromic displays a wavelength-dependent competition between fluorescence and photocyclization assigned to a red-shifted absorption of the fluorescing conformer compared to the absorption of the photoreactive conformer. 相似文献
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Yael Nissinkorn Naama Lahav‐Mankovski Dr. Aharon Rabinkov Dr. Shira Albeck Dr. Leila Motiei Dr. David Margulies 《Chemistry (Weinheim an der Bergstrasse, Germany)》2015,21(45):15981-15987
A methodology for creating fluorescent molecular sensors that respond to changes that occur on the surfaces of specific proteins is presented. This approach, which relies on binding cooperatively between a specific His‐tag binder and a nonspecific protein‐surface receptor, enabled the development of a sensor that can track changes on the surface of a His‐tag‐labeled calmodulin (His‐CaM) upon interacting with metal ions, small molecules, and protein binding partners. The way this approach was used to detect dephosphorylation of an unlabeled calmodulin‐dependent protein kinase II (CaMKII), and the binding of Bax BH3 to His‐tagged B‐cell lymphoma 2 (Bcl‐2) protein is also presented. 相似文献
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Dr. Sina Berndl Stoichko D. Dimitrov Dr. Florian Menacher Prof. Dr. Torsten Fiebig Prof. Hans‐Achim Wagenknecht 《Chemistry (Weinheim an der Bergstrasse, Germany)》2016,22(7):2386-2395
By using (S)‐2‐amino‐1,3‐propanediol as a linker, thiazole orange (TO) was incorporated in a dimeric form into DNA. The green fluorescence (λ=530 nm) of the intrastrand TO dimer is quenched, whereas the interstrand TO dimer shows a characteristic redshifted orange emission (λ=585 nm). Steady‐state optical spectroscopic methods reveal that the TO dimer fluorescence is independent of the sequential base contexts. Time‐resolved pump–probe measurements and excitation spectra reveal the coexistence of conformations, including mainly stacked TO dimers and partially unstacked ones, which yield exciton and excimer contributions to the fluorescence, respectively. The helicity of the DNA framework distorts the excitonic coupling. In particular, the interstrand TO dimer could be regarded as an excitonically interacting base pair with fluorescence readout for DNA hybridization. Finally, the use of this fluorescent readout was representatively demonstrated in molecular beacons. 相似文献
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Xueying Wang Lei Wang Xinping Lin Xiaobing Yang Wujun Liu Zongbao K. Zhao 《Applied biochemistry and biotechnology》2018,185(1):81-90
Directed evolution-based protein engineering usually generates large library contained insoluble mutants because of structural disturbance by mutation. To reduce the workload and costs, it is crucial to identify and eliminate those insoluble variants prior to dedicated analysis. Here, we demonstrate a method to visualize soluble protein mutants by using monomeric red fluorescent protein (mRFP) as a fusion tag. A plasmid was devised to express nicotinic acid mononucleotide adenylyltransferase (NadD) fused with a GGGS-linked mRFP tag at the C-terminus. The plasmid was subjected to site saturation mutagenesis within the nadD gene, used to transform Escherichia coli DH10B competent cells, leading to colonies with different red intensities. It was found that the fluorescence intensity of the cell culture correlated positively with the content of NadD-mRFP mutant in the supernatant. Mutation at position 132 led to a library of which most colonies lost the red phenotype, indicating that the position had a key role for proper protein folding. Similarly, mRFP enabled identification of soluble mutants of other enzymes including 1-deoxy-D-xylulose-5-phosphate reductoisomerase and phosphite dehydrogenase. These data suggested that mRFP can serve as a fusion reporter for visualizing soluble protein mutants to facilitate more efficient library screening in directed evolution. 相似文献
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Taylor D. Krueger Dr. Longteng Tang Dr. Liangdong Zhu Isabella L. Breen Prof. Dr. Rebekka M. Wachter Prof. Dr. Chong Fang 《Angewandte Chemie (Weinheim an der Bergstrasse, Germany)》2020,132(4):1661-1669
The molecular mechanisms for the photoconversion of fluorescent proteins remain elusive owing to the challenges of monitoring chromophore structural dynamics during the light-induced processes. We implemented time-resolved electronic and stimulated Raman spectroscopies to reveal two hidden species of an engineered ancestral GFP-like protein LEA, involving semi-trapped protonated and trapped deprotonated chromophores en route to photoconversion in pH 7.9 buffer. A new dual-illumination approach was examined, using 400 and 505 nm light simultaneously to achieve faster conversion and higher color contrast. Substitution of UV irradiation with visible light benefits bioimaging, while the spectral benchmark of a trapped chromophore with characteristic ring twisting and bridge-H bending motions enables rational design of functional proteins. With the improved H-bonding network and structural motions, the photoexcited chromophore could increase the photoswitching-aided photoconversion while reducing trapped species. 相似文献
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次氯酸(HCl O)是生物体内重要的活性氧(ROS)之一,在人类免疫功能系统中扮演着重要的角色,有助于对入侵细菌和病原体进行破坏。本文设计并合成了基于香豆素为母体单元的比率型次氯酸荧光探针。研究结果表明,该探针对次氯酸识别显示出较高的选择性,检测线低至12 mol/L,荧光响应可在5 s内迅速完成,并伴随着溶液颜色由无色转变为黄绿色。其它常见的阴离子及氧化型物质对次氯酸检测均无干扰。此外,高分辨率质谱、荧光光谱和紫外可见光谱变化共同证实了该探针对次氯酸的检测机制为次氯酸对探针氧化水解。 相似文献
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《Chemistry & biology》2014,21(10):1402-1414
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David Margulies Dr. Andrew D. Hamilton Prof. 《Angewandte Chemie (International ed. in English)》2009,48(10):1771-1774
Sniffing out proteins : Fluorescent DNA G‐quadruplexes have been used for building versatile signaling receptors for proteins in a single solution. Introducing a protein sample to the ensemble results in a unique emission signature for unambiguous identification (see scheme, R=fluorophore). The self‐assembled, pattern‐based protein detection systems are easily fabricated, have the potential for high‐throughput operations, and have the ability to handle small protein samples.
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Highly Fluorescent Green Fluorescent Protein Chromophore Analogues Made by Decorating the Imidazolone Ring 下载免费PDF全文
Sara Gutiérrez David Martínez‐López Dr. María Morón Dr. David Sucunza Dr. Diego Sampedro Dr. Alberto Domingo Dr. Antonio Salgado Prof. Juan J. Vaquero 《Chemistry (Weinheim an der Bergstrasse, Germany)》2015,21(51):18758-18763
The synthesis and photophysical behavior of an unexplored family of green fluorescent protein (GFP)‐like chromophore analogues is reported. The compound (Z)‐4‐(4‐hydroxybenzylidene)‐1‐propyl‐2‐(propylamino)‐1H‐imidazol‐5(4 H)‐one (p‐HBDNI, 2 a ) exhibits significantly enhanced fluorescence properties relative to the parent compound (Z)‐5‐(4‐hydroxybenzylidene)‐2,3‐dimethyl‐3,5‐dihydro‐4H‐imidazol‐4‐one (p‐HBDI, 1 ). p‐HBDNI was considered as a model system and the photophysical properties of other novel 2‐amino‐3,5‐dihydro‐4H‐imidazol‐4‐one derivatives were evaluated. Time‐dependent DFT calculations were carried out to rationalize the results. The analogue AIDNI ( 2 c ), in which the 4‐hydroxybenzyl group of p‐HBDNI was replaced by an azaindole group, showed improved photophysical properties and potential for cell staining. The uptake and intracellular distribution of 2 c in living cells was investigated by confocal microscopy imaging. 相似文献
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以香豆素343和2-苯并噻唑乙腈为原料,合成了香豆素343-亚硫酸根离子探针(Coumarin 343-SO2),并用1H NMR、13C NMR、MS和HRMS等技术手段对合成的化合物进行了表征。 该探针与亚硫酸根离子(SO32-)发生亲核加成反应后,阻断了苯并噻唑与香豆素的共轭结构,从而引起荧光强度的变化,达到检测亚硫酸根离子的目的。 此探针对SO32-具有响应快、高灵敏度、高选择性及检测限低至0.08 μmol/L的特点,其它常见的阴离子及还原性物质对SO32-的检测均无干扰。 此外,该探针具有良好的细胞膜通透性,可用于活细胞中对SO32-进行荧光成像。 相似文献
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Understanding the composition, function and regulation of complex cellular systems requires tools that quantify the expression of multiple proteins at their native cellular context. Here, we report a highly sensitive and accurate protein in situ profiling approach using off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this method, protein targets are stained with horseradish peroxidase (HRP) conjugated antibodies and CFT. Subsequently, the fluorophores are efficiently cleaved by mild chemical reagents, which simultaneously deactivate HRP. Through reiterative cycles of protein staining, fluorescence imaging, fluorophore cleavage, and HRP deactivation, multiplexed protein quantification in single cells in situ can be achieved. We designed and synthesized the high-performance CFT, and demonstrated that over 95% of the staining signals can be erased by mild chemical reagents while preserving the integrity of the epitopes on protein targets. Applying this method, we explored the protein expression heterogeneity and correlation in a group of genetically identical cells. With the high signal removal efficiency, this approach also enables us to accurately profile proteins in formalin-fixed paraffin-embedded (FFPE) tissues in the order of low to high and also high to low expression levels. 相似文献
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An Upconversion Nanoparticle with Orthogonal Emissions Using Dual NIR Excitations for Controlled Two‐Way Photoswitching 下载免费PDF全文
Jinping Lai Yixiao Zhang Nicholas Pasquale Ki‐Bum Lee 《Angewandte Chemie (International ed. in English)》2014,53(52):14419-14423
Developing multicolor upconversion nanoparticles (UCNPs) with the capability of regulating their emission wavelengths in the UV to visible range in response to external stimuli can offer more dynamic platforms for applications in high‐resolution bioimaging, multicolor barcoding, and driving multiple important photochemical reactions, such as photoswitching. Here, we have rationally designed single‐crystal core–shell‐structured UCNPs which are capable of orthogonal UV and visible emissions in response to two distinct NIR excitations at 808 and 980 nm. The orthogonal excitation–emission properties of such UCNPs, as well as their ability to utilize low‐power excitation, which attenuates any local heating from the lasers, endows the UCNPs with great potential for applications in materials and biological settings. As a proof of concept, the use of this UCNP for the efficient regulation of the two‐way photoswitching of spiropyran by using dual wavelengths of NIR irradiation has been demonstrated. 相似文献
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基于聚合酶反应和发夹型核酸适体的蛋白质荧光检测新方法 总被引:1,自引:0,他引:1
设计合成了一种发夹型核酸适体(Aptamer), 结合聚合酶反应建立了蛋白质荧光分析新方法. 该核酸适体同时作为蛋白质配体和聚合反应模板, 与靶蛋白特异结合后, 其构象发生了变化, 启动聚合反应, 从而在未直接标记核酸适体的情况下, 通过监测聚合反应进程来检测蛋白质的浓度. 采用该方法检测凝血酶的线性范围为0.5~8 nmol/L, 检测下限为0.5 nmol/L, 为蛋白质检测提供了一种简便快速的非直接标记的荧光分析方法, 有望在蛋白质组学的研究中得到广泛的应用. 相似文献
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绿色荧光蛋白质(GFP)的发现、表达和发展 总被引:6,自引:0,他引:6
吴世康 《影像科学与光化学》2009,27(1):69-78
本文简要地介绍了2008年Nobel化学奖——绿色荧光蛋白质的主要学术内容,包括绿色荧光蛋白质的发现、发展和工作的重要意义.类-绿色荧光蛋白质可用于在时间和空间上监视越来越多的活细胞中的现象和机制,如基因表达、蛋白质的定位和动态学等.因此可以说,绿色荧光蛋白质的发现是联系到生物科学上的一次技术革命. 相似文献