Concerning the Thermal Diastereomerization of the Green Fluorescent Protein Chromophore

Summary. Two model compounds for the green fluorescent protein chromophore were prepared. One of them incorporates the natural 4-hydroxybenzylidene group of the natural tyrosin derived chromophore, the other one bears a methyl group instead of the hydroxy group. Whereas the photochemically prepared (E)-diastereomer of the first compound very effectively reverted thermally (room temperature) to the thermodynamically stable (Z)-diastereomer, the (E)-diastereomer of the second derivative proved to be stable even at elevated temperatures for more than a day. This finding can be rationalized by constructing the appropriate resonance structures showing that only in the first case an effective delocalization enables partial single bond character of the benzylidene double bond. From the standpoint of chemical etiology, only Nature’s choice of the tyrosin derived chromophore of the green fluorescent protein provides an efficient radiationless thermal relaxation channel for the unwanted photo-diastereomerization product formed after excitation besides the dominating fluorescence channel of its chromophore.     

绿色荧光蛋白

绿色荧光蛋白是46多年前从多管水母体内发现的,它可以在蓝光或紫外光激发下发射绿光.由于它稳定的结构和光物理性质,又易于在细胞内表达,近些年作为标记物已经被广泛地应用于生命科学领域.本文简要介绍了水母发光蛋白与绿色荧光蛋白的关系、绿色荧光蛋白的结构、发色团的形成、发光机制、变异体以及它的特点和应用.     

绿色荧光蛋白在细胞分析中的应用

本文对绿色荧光蛋白的光谱特性及其在细胞分析中的应用进行了评述。     

绿色荧光蛋白标记检测枯草芽孢杆菌在水体中的存活动态

基于生物-化学协同控制植物病害的原理,构建了一种由枯草芽孢杆菌和烯酰吗啉组成的对辣椒等作物疫病具有较好的防治效果的菌药合剂(DMBS)。按照农药降解研究基本规则,采用绿色荧光蛋白(greenfluorescent protein,GFP)标记技术和细菌学研究方法研究了DMBS在去离子水、地下水、自来水、河水和雨水中的降解动态。结果表明,GFP标记可以用于枯草芽孢杆菌在5种水环境中的存活检测。在(25±1)℃条件下,枯草芽孢杆菌菌剂和DMBS中的枯草芽孢杆菌数量主要表现为前12 d迅速下降,此后则随时间的延长在一定的范围内呈变动的上升或下降趋势。在(50±1)℃灭菌和不灭菌的条件下,均表现为前12 d迅速下降,12 d后趋于稳定或缓慢下降。枯草芽孢杆菌在5种水中的降解速度较慢,在(25±1)℃和(50±1)℃条件下存放86 d后,其含量均在104cfu/mL以上。培养温度和灭菌条件对枯草芽孢杆菌在不同水体中存活动态有一定的影响,菌药合剂中的烯酰吗啉对该菌的存活则没有显著影响。     

绿色荧光蛋白质(GFP)的发现、表达和发展

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.     

Highly Fluorescent Green Fluorescent Protein Chromophore Analogues Made by Decorating the Imidazolone Ring

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.     

含绿色荧光蛋白发色团双自由基分子的非线性光学性质

采用密度泛函理论(DFT)方法研究了系列含绿色荧光蛋白发色团双自由基分子光学异构体的几何结构、极化率(αs)和第一超极化率(βtot).结果表明,引入电子给受体取代基使分子的极化率增大,而对第一超极化率有不同影响.对于光照前的反式结构,引入电子受体βtot值增加,且βtot值随取代基吸电子能力的增强而增大;引入电子给体βtot值降低,且βtot值随取代基给电子能力的增强而减小.当分子变成相应的顺式结构时,其βtot值变化趋势与反式结构的结果正好相反.光异构化前后分子的βtot值变化不同,引入电子受体使顺式结构的βtot值比反式结构的小,其中―NO2使顺式结构的βtot值减小为反式结构的1/6;引入电子给体使反式结构的βtot值比顺式结构的小,其中―NH2使反式结构的βtot值减小为顺式结构的1/6.从而,光异构化起到调节非线性光学(NLO)响应的作用.     

荧光蛋白标记/高效液相色谱分析枯草芽孢杆菌-烯酰吗啉菌药合剂

生物和化学组分构建的菌药合剂是新农药制剂发展的热点,其中生物活体和化学组分的准确、快速检测方法备受关注。本实验应用绿色荧光蛋白标记和高效液相色谱技术研究了枯草芽孢杆菌-烯酰吗啉菌药合剂在54±2℃贮存14d前后生防菌株和化学组分含量的变化。结果表明:绿色荧光蛋白标记和高效液相色谱技术联用,可以用于该菌药合剂质量检测和热贮实验中混剂的贮存稳定性监控;菌药合剂在热贮实验过程中的组分含量变化在允许变化范围之内。     

绿色荧光蛋白质——2008年诺贝尔化学奖成果介绍

介绍了2008年诺贝尔化学奖得主下村修、马丁.查尔菲和钱永健3位科学家在绿色荧光蛋白质的发现和开发方面的卓越贡献,阐述了绿色荧光蛋白质的结构、性质以及应用等。     

Metal-Induced Fluorescence Quenching of Photoconvertible Fluorescent Protein DendFP

Sensitive and accurate detection of specific metal ions is important for sensor development and can advance analytical science and support environmental and human medical examinations. Fluorescent proteins (FPs) can be quenched by specific metal ions and spectroscopically show a unique fluorescence-quenching sensitivity, suggesting their potential application as FP-based metal biosensors. Since the characteristics of the fluorescence quenching are difficult to predict, spectroscopic analysis of new FPs is important for the development of FP-based biosensors. Here we reported the spectroscopic and structural analysis of metal-induced fluorescence quenching of the photoconvertible fluorescent protein DendFP. The spectroscopic analysis showed that Fe²⁺, Fe³⁺, and Cu²⁺ significantly reduced the fluorescence emission of DendFP. The metal titration experiments showed that the dissociation constants (K_d) of Fe²⁺, Fe³⁺, and Cu²⁺ for DendFP were 24.59, 41.66, and 137.18 μM, respectively. The tetrameric interface of DendFP, which the metal ions cannot bind to, was analyzed. Structural comparison of the metal-binding sites of DendFP with those of iq-mEmerald and Dronpa suggested that quenchable DendFP has a unique metal-binding site on the β-barrel that does not utilize the histidine pair for metal binding.     

Green fluorescent protein for in situ synthesis of highly uniform Au nanoparticles and monitoring protein denaturation

Purified recombinant green fluorescent protein (GFP) expressed in E. coli was used for single-step synthesis of gold nanoparticles (Au NPs) with extraordinary size specificity in aqueous medium. The fluorescence of GFP offered a probe for concomitant changes in the protein during the course of synthesis, in addition to the monitoring of the time-dependent formation of Au NPs by the surface plasmon resonance. Reaction of AuCl⁻₄ with the protein produced spherical Au NPs having diameters ranging from 5–70 nm. Remarkably, addition of 1.0×10⁻⁵ M AgNO₃ in the medium produced uniform spherical Au NPs with particle diameter of 2.2±0.5 nm. Fluorescence spectroscopic measurements suggest that during synthesis of Au NPs in absence of AgNO₃, partial denaturation of the protein occurred resulting in the lowering of fluorescence intensity. On the other hand, when the NPs were synthesized in the presence of AgNO₃ complete denaturation of the protein with complete loss of fluorescence could be observed, which was further confirmed by native and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). However, use of AgNO₃ only resulted neither in the formation of NPs nor had any significant effect on the fluorescence of GFP.     

Concerted Asynchronous Hula‐Twist Photoisomerization in the S65T/H148D Mutant of Green Fluorescent Protein

Fluorescence emission of wild‐type green fluorescent protein (GFP) is lost in the S65T mutant, but partly recovered in the S65T/H148D double mutant. These experimental findings are rationalized by a combined quantum mechanics/molecular mechanics (QM/MM) study at the QM(CASPT2//CASSCF)/AMBER level. A barrierless excited‐state proton transfer, which is exclusively driven by the Asp148 residue introduced in the double mutant, is responsible for the ultrafast formation of the anionic fluorescent state, which can be deactivated through a concerted asynchronous hula‐twist photoisomerization. This causes the lower fluorescence quantum yield in S65T/H148D compared to wild‐type GFP. Hydrogen out‐of‐plane motion plays an important role in the deactivation of the S65T/H148D fluorescent state.     

Sensing Protein Surfaces with Targeted Fluorescent Receptors

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.     

健那绿的荧光光谱特性研究

本文研究了酸度、离子强度、介质等因素对健那绿荧光光谱的影响。发现随着体系酸度的增加,健那绿的荧光强度呈增强趋势,但在pH>4.54后,体系的荧光强度会发生波动。体系的离子强度不仅会影响健那绿的荧光强度,也会使发射波长红移。体系中乙醇的存在会使健那绿的荧光强度增强。测得健那绿在水和乙醇中的荧光量子产率分别为0.10～0.26和0.25～0.50,荧光偏振分别为0.11和0.04。     

Fluorescent Light‐up Probe for the Detection of Protein Aggregates

A fluorescent dye was decorated with water‐soluble pyridinium groups in order to be applied in the detection of cyclodextrins or DNA. The dye displays an enhancement of its emission intensity when the internal rotations are restricted due to the formation of an inclusion complex with cyclodextrins or upon interaction with DNA. In vivo, the fluorescent probe can stain protein aggregates with a selectivity comparable to the widely used Proteostat®.     

蛋白质分子荧光探针研究及其应用新进展

人体中多达10万种以上的蛋白质结构,功能千差万别,形成了生命的多样性和复杂性。在分子水平上分析和识别蛋白质对生命科学研究具有重要的理论和实践意义。本文综述了各种蛋白质分子荧光探针在蛋白质分析方面的应用,并展望了此类荧光探针的发展趋势和应用前景。引用文献60篇。     

Mechanically Modulating the Photophysical Properties of Fluorescent Protein Biocomposites for Ratio‐ and Intensiometric Sensors

Mechanically sensitive biocomposites comprised of fluorescent proteins report stress through distinct pathways. Whereas a composite containing an enhanced yellow fluorescent protein (eYFP) exhibited hypsochromic shifts in its fluorescence emission maxima following compression, a composite containing a modified green fluorescent protein (GFPuv) exhibited fluorescence quenching under the action of mechanical force. These ratio‐ and intensiometric sensors demonstrate that insights garnered from disparate fields (that is, polymer mechanochemistry and biophysics) can be harnessed to guide the rational design of new classes of biomechanophore‐containing materials.