荧光光谱在蛋白质分子构象研究中的应用*

荧光光谱法是研究蛋白质在水溶液中分子构象的一种有效方法。文章综述了常见的蛋白质荧光光谱的研究方法, 并介绍了几种荧光光谱新技术。     

Protein Enhanced Near-Infrared Fluorescence of AuNPs and Its Application for Protein Determination

The 15～25 nm water soluble and stable gold nanoparticles were synthesized and studied for their spectral properties and interactions with proteins. Results showed that 15 nm gold nanoparticles can emit near infrared fluorescence with an emission peak of 811.2 nm under the excitation of 538 nm. The study also showed that proteins can obviously enhance the near infrared fluorescence intensity of gold nanoparticles. Under the optimized conditions, there is a linear relationship between the fluorescence intensity enhancement of the system and the concentrations of the proteins, which can be used in a new method for the determination of trace proteins. The mechanisms of the interaction and the fluorescence enhancement of the nano-gold-protein system were also studied.     

蛋白质固体表面荧光分析法研究

本文以滤纸为基质研究曙红Y与牛血清蛋(BSA)白作用,建立了蛋白质的固体表面荧光分析法。实验证明,本方法具有操作简便、灵敏度高(检出限0.15mg·L-1)、线性范围宽(0.15～50mg·L-1)、取样量少(6μL/spot)等特点,对合成样品中的牛血清蛋白(BSA)测定结果令人满意。     

Revisiting the Rate-Limiting Step of the ANS–Protein Binding at the Protein Surface and Inside the Hydrophobic Cavity

8-Anilino-1-naphthalenesulfonic acid (ANS) is used as a hydrophobic fluorescence probe due to its high intensity in hydrophobic environments, and also as a microenvironment probe because of its unique ability to exhibit peak shift and intensity change depending on the surrounding solvent environment. The difference in fluorescence can not only be caused by the microenvironment but can also be affected by the binding affinity, which is represented by the binding constant (K). However, the overall binding process considering the binding constant is not fully understood, which requires the ANS fluorescence binding mechanism to be examined. In this study, to reveal the rate-limiting step of the ANS–protein binding process, protein concentration-dependent measurements of the ANS fluorescence of lysozyme and bovine serum albumin were performed, and the binding constants were analyzed. The results suggest that the main factor of the binding process is the microenvironment at the binding site, which restricts the attached ANS molecule, rather than the attractive diffusion-limited association. The molecular mechanism of ANS–protein binding will help us to interpret the molecular motions of ANS molecules at the binding site in detail, especially with respect to an equilibrium perspective.     

Comparing the Affinities of Flavonoid Isomers with Protein by Fluorescence Spectroscopy

Abstract

Dietary flavonoids can be detected in plasma as protein‐bound conjugates. Flavonoids–protein interaction is expected to modulate the bioavailability of flavonoids. In this work, the binding flavonoid isomers (galangin, baicalein, apigenin, and genistein; MW=270.25) and B‐ring hydroxylation flavonols (galangin, kaempferol, quercetin, and myricetin, which share the same structure on the A and C rings but have 0, 1, 2, and 3 moieties of ‐OH on the B‐ring, respectively) to protein were investigated by fluorescence quenching method. The apparent binding constants (K _a ) of were flavonoid isomers determined as: flavones (10⁶–10⁷ L mol^?1)>isoflavone≈flavonol (10⁵ L mol^?1). For B‐ring hydroxylation flavonols, the binding affinity increased with increasing number of hydroxyl groups on the B‐ring. The binding constants (K _a ) were determined as follows: myricetin>quercetin>kaempferol>galangin.     

蛋白质微阵列芯片技术及其在抗体筛选中的应用

以兔IgG为模式蛋白质,对其在醛基修饰玻片表面的固定浓度、固定时间和温度等条件进行了优化,结果表明：在室温下,当固定蛋白质的浓度为1g／L、固定时间为4h时,可获得理想的蛋白质固定效果;蛋白质的定量检测范围为1μg／L～10mg／L。按优化的蛋白质微阵列芯片制作条件将规模化制备的抗体制作成抗体微阵列芯片,通过与荧光标记的人球蛋白和人白蛋白的相互作用,实现了对不同抗体株抗球蛋白和抗白蛋白活性的快速筛选与比较。     

Red-Shifted Substrates for FAST Fluorogen-Activating Protein Based on the GFP-Like Chromophores

A genetically encoded fluorescent tag for live cell microscopy is presented. This tag is composed of previously published fluorogen-activating protein FAST and a novel fluorogenic derivative of green fluorescent protein (GFP)-like chromophore with red fluorescence. The reversible binding of the novel fluorogen and FAST is accompanied by three orders of magnitude increase in red fluorescence (580–650 nm). The proposed dye instantly stains target cellular proteins fused with FAST, washes out in a minute timescale, and exhibits higher photostability of the fluorescence signal in confocal and widefield microscopy, in contrast with previously published fluorogen:FAST complexes.     

Synchronous Fluorescence Determination of Protein with Functional Organic Nanoparticles

A new method for detecting protein using a synchronous fluorescence scan technique with a novel composite organic nanoparticle has been developed. The novel functional organic nanoparticle, dodecyl benzene sulfonic acid sodium salt (DBSS)-capped nano-anthracene, is easily prepared by re-precipitation under ultrasonic agitation. The surface of the fluorescent organic nanoparticle was covered with functional groups, which made these nanoparticles water-soluble, stable, and biocompatible. Compared to single organic fluorophores, these nanoparticles are brighter, more resistant to photobleaching, and do not suffer from blinking. We developed a sensitive synchronous fluorescence method for the rapid determination of proteins. With Δλ = 60 nm, maximum and constant synchronous fluorescence is produced at 339 nm. Under optimal conditions, the linear range is 0.04–13.0 μg mL⁻¹ for γ-globulin (γ-G), 0.1–11 μg mL⁻¹ for human serum albumin (HSA), and 0.1–11 μg mL⁻¹ for bovine serum albumin (BSA), respectively. The method is characterized by reproducibility, excellent accuracy and few interfering substances. The proposed method was applied to the determination of human serum samples collected from a hospital, and the results were in good agreement with those reported by the hospital, indicating that the method presented here is not only sensitive and simple, but also reliable and suitable for practical application.     

Spectroscopic Investigation of the Structure of a Protein Adsorbed on a Hydrophobic Latex

Spectroscopic methods provide a powerful tool for investigating the structural properties of immobilized proteins. For that purpose, there was a strong need for a hydrophobic particle that allows determination of the adsorbed protein conformation by fluorescence and circular dichroism. Among the various hydrophobic suspensions that are available, perfluoro-alkoxy fluoro carbon Teflon latex satisfies the requirements of low light absorption and scattering. As an example, preliminary results of structural changes of a proteolytic enzyme dissolved in aqueous solution and in the adsorbed state are given.     

Confined Water in Amyloid‐Competent Oligomers of the Prion Protein

Conformational switching of the prion protein into the abnormal form involves the formation of (obligatory) molten‐oligomers that mature into ordered amyloid fibrils. The role of water in directing the course of amyloid formation remains poorly understood. Here, we show that the mobility of the water molecules within the on‐pathway oligomers is highly retarded. The water relaxation time within the oligomers was estimated to be ≈1 ns which is about three orders of magnitude slower than the bulk water and resembles the characteristics of (trapped) nano‐confined water. We propose that the coalescence of these obligatory oligomers containing trapped water is entropically favored because of the release of ordered water molecules in the bulk milieu and results in the sequestration of favorable inter‐chain amyloid contacts via nucleated conformational conversion. The dynamic role of water in protein aggregation will have much broader implications in a variety of protein misfolding diseases.     

Conformational Stability of Bovine Serum Albumin in Aqueous Amides: A Further Insight into the Mechanism of Urea Acting on the Protein

The binding distances of fluorescein to bovine serum albumin (BSA) in formamide‐water and N,N‐dimethyl‐ formamide‐water mixtures were determined by fluorescence quenching method and compared with the values in urea‐water mixtures in our previous work. The results, together with the analysis of fluorescence spectra, were utilized to probe the conformational stability of protein in aqueous amides, providing a further insight into the mechanism of urea acting on protein. The spectral properties of BSA showed significant difference in the aqueous solutions of the three kinds of amide and indicated that both NH₂ group and C=O group could form hydrogen bond with the protein, serving as donor and acceptor, respectively. However, the results revealed that the multiple hydrogen bonds of NH₂ group with back bond and hydrophilic side chains of the protein played a key role in the nonspecific urea‐mediated network of intramolecular interaction due to its higher hydrogen bonding capability compared to C=O group.     

蛋白微阵列免疫分析法用于海洋致病菌的定量检测

建立了一种用于水体中副溶血弧菌、河弧菌和大肠杆菌检测定量的蛋白微阵列免疫分析法。以Cy3标记免疫球蛋白(IgG)为探针,蛋白芯片为载体,对孵育反应的IgG浓度、反应时间和温度等条件进行了优化,结果表明:当IgG浓度为0.1g/L在37℃下孵育60min,可以获得理想的检测效果;在优化条件下,本方法中副溶血弧菌、河弧菌和大肠杆菌的检出限分别为9.9×104个/mL、9.3×104个/mL和3.9×105个/mL;将本方法与ELISA进行了比较,结果表明,本方法具有快速、操作便捷、高通量检测等特点;并将本方法应用于海水中这3种致病菌的快速、准确地定量检测。     

Protein scissors: Photocleavage of proteins at specific locations

Site-specific photocleavage of hen egg lysozyme and bovine serum albumin (BSA) by N-(L-phenylalanine)-4(1-pyrene)butyramide (Py-Phe) is investigated in detail with respect to its efficiency, dependence on oxygen, and radical quenchers. Binding of Py-Phe to BSA follows abiphasic process with two binding sites per protein. The photocleavage was achieved upon irradiating a mixture of protein, Py-Phe and Co(III)hexammine (CoHA) at 344 nm. No protein cleavage was observed in the absence of Py-Phe, or CoHA, or light. Photocleavage of BSA was inhibited by degassing or by the addition of radical quenchers such as ethanol. In addition, the photoreaction was quenched by electron donors such as ethanolamine. This result was corroborated by the flash photolysis studies where the cation radical derived from the probe is also quenched by ethanolamine with an equivalent rate constant. Quenching of the singlet excited state of Py-Phe by CoHA followed by the reaction of the resulting pyrenyl cation radical with the protein backbone is the suggested mechanism of protein cleavage. The origin of the specificity of photocleavage is discussed and specificity is valuable in targeting desired sites of proteins with small molecules.     

Protein determination using methylene blue in a synchronous fluorescence technique

A new method for detecting protein by synchronous fluorescence enhancement was developed, based on the combination of near infrared (NIR) fluorescence and the dedimerization phenomenon of methylene blue (MB). Under analytical conditions, there are linear relationships between the enhancing extent of synchronous fluorescence of MB-sodium dodecyl benzene sulfonate (SDBS)-protein at 667 nm and the concentration of protein in the range of 8.0 × 10⁻⁸-4.0 × 10⁻⁵ g mL⁻¹ for bovine serum albumin (BSA), 1.0 × 10⁻⁷-3.5 × 10⁻⁵ g mL⁻¹ for egg albumin (EA). The detection limits (S/N = 3) of BSA and EA are 8.9 ng mL⁻¹ and 10.0 ng mL⁻¹, respectively. The fluorescence enhancement mechanism is discussed in detail. Results from multiple techniques indicate that the fluorescence enhancement of the system originates from the hydrophobic microenvironment provided by BSA and SDBS, and the formation of an MB-SDBS-BSA complex, as well as the deaggregation of some MB dimer.     

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.     

Color Tuning of Fluorogens for FAST Fluorogen-Activating Protein

Using benzylidene imidazolone core, we created a panel of color-shifted fluorogenic ligands for FAST protein without compromise to the binding efficiency and the utility for live-cell protein labeling. This study highlights the potential of benzylidene imidazolones derivatives for rapid expansion of a pallet of live-cell fluorogenic labeling tools.     

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.     

Structural Changes in a Protein Fragment from Abalone Shell during the Precipitation of Calcium Carbonate

Mineralized tissues grow through biologically controlled processes in which specific macromolecules are involved. Some of these molecules, which are present in very low concentrations and are difficult to localize and characterize, become entrapped inside the mineralized tissue. Herein, a protein fragment, GP, which was obtained by the alkaline digestion of the green sheet of the abalone shell, is used as a probe to study the changes in molecular structure that occur during the precipitation of calcium carbonate. This important goal was achieved by exploiting a fluorescent tag in GP. The experimental results that were obtained by using spectroscopic‐, chromatographic‐, and microscopic techniques indicate that GP controls the precipitation kinetics and morphology of calcium carbonate crystals, and that it only undergoes structural reorganization when entrapped inside calcium carbonate crystals. To the best of our knowledge, this report represents one of the first studies on the conformational changes of a protein fragment that is involved in biomineralization processes on moving from the solution phase into the mineral phase.     

A Water‐Soluble Conjugated Polymer for Protein Identification and Denaturation Detection

Rapid and sensitive methods to detect proteins and protein denaturation have become increasingly needful in the field of proteomics, medical diagnostics, and biology. In this paper, we have reported the synthesis of a new cationic water‐soluble conjugated polymer that contains fluorene and diene moieties in the backbone ( PFDE ) for protein identification by sensing an array of PFDE solutions in different ionic strengths using the linear discriminant analysis technique (LDA). The PFDE can form complexes with proteins by electrostatic and/or hydrophobic interactions and exhibits different fluorescence response. Three main factors contribute to the fluorescence response of PFDE , namely, the net charge density on the protein surface, the hydrophobic nature of the protein, and the metalloprotein characteristics. The denaturation of proteins can also be detected using PFDE as a fluorescent probe. The interactions between PFDE and proteins were also studied by dynamic light scattering (DLS) and isothermal titration microcalorimetry (ITC) techniques. In contrast to other methods based on conjugated polymers, the synthesis of a series of quencher or dye‐labeled acceptors or protein substrates has been avoided in our method, which significantly reduces the cost and the synthetic complexity. Our method provides promising applications on protein identification and denaturation detection in a simple, fast, and label‐free manner based on non‐specific interaction‐induced perturbation of PFDE fluorescence response.