Precise Quantification of Molybdate In Vitro by the FRET-Based Nanosensor ‘MolyProbe’

Molybdenum (Mo) is an essential trace element in all kingdoms of life. Mo is bioavailable as the oxyanion molybdate and gains biological activity in eukaryotes when bound to molybdopterin, forming the molybdenum cofactor. The imbalance of molybdate homeostasis results in growth deficiencies or toxic symptoms within plants, fungi and animals. Recently, fluorescence resonance energy transfer (FRET) methods have emerged, monitoring cellular and subcellular molybdate distribution dynamics using a genetically encoded molybdate-specific FRET nanosensor, named MolyProbe. Here, we show that the MolyProbe system is a fast and reliable in vitro assay for quantitative molybdate determination. We added a Strep-TagII affinity tag to the MolyProbe protein for quick and easy purification. This MolyProbe is highly stable, resistant to freezing and can be stored for several weeks at 4 °C. Furthermore, the molybdate sensitivity of the assay peaked at low nM levels. Additionally, The MolyProbe was applied in vitro for quantitative molybdate determination in cell extracts of the plant Arabidopsis thaliana, the fungus Neurospora crassa and the yeast Saccharomyces cerevisiae. Our results show the functionality of the Arabidopsis thaliana molybdate transporter MOT1.1 and indicate that FRET-based molybdate detection is an excellent tool for measuring bioavailable Mo.     

Metal ion induced FRET On-Off in naphthyl-pyrenyl pendent tetrahomodioxacalix[4]arene

A novel tetrahomodioxacalix[4]arene (7) bearing both naphthyl- and pyrenyl-amide pendants was synthesized as FRET-based fluorometric sensor for Cu²⁺ ion. Intramolecular FRET from the naphthalene emission to the pyrene absorption affords Cu²⁺ ion selectivity over other metal ions. Upon addition of Cu²⁺ ion, the complex solution of 7 gave a significantly decreased pyrene acceptor emission along with an enhancement of naphthalene donor emission via FRET On-Off event.     

Förster Resonance Energy Transfer Investigations Using Quantum‐Dot Fluorophores

F?rster resonance energy transfer (FRET), which involves the nonradiative transfer of excitation energy from an excited donor fluorophore to a proximal ground-state acceptor fluorophore, is a well-characterized photophysical tool. It is very sensitive to nanometer-scale changes in donor-acceptor separation distance and their relative dipole orientations. It has found a wide range of applications in analytical chemistry, protein conformation studies, and biological assays. Luminescent semiconductor nanocrystals (quantum dots, QDs) are inorganic fluorophores with unique optical and spectroscopic properties that could enhance FRET as an analytical tool, due to broad excitation spectra and tunable narrow and symmetric photoemission. Recently, there have been several FRET investigations using luminescent QDs that focused on addressing basic fundamental questions, as well as developing targeted applications with potential use in biology, including sensor design and protein conformation studies. Herein, we provide a critical review of those developments. We discuss some of the basic aspects of FRET applied to QDs as both donors and acceptors, and highlight some of the advantages offered (and limitations encountered) by QDs as energy donors and acceptors compared to conventional dyes. We also review the recent developments made in using QD bioreceptor conjugates to design FRET-based assays.     

Interaction of Squid (Dosidicus giga) Mantle Protein with a Mixtures of Potato and Corn Starch in an Extruded Snack,as Characterized by FTIR and DSC

The majority of snacks expanded by extrusion (SEE) are made with vegetable sources, to improve their nutritional content; it has been proposed to incorporate squid (Dosidicus gigas), due to its high protein content, low price and high availability. However, the interaction of proteins of animal origin with starch during extrusion causes negative effects on the sensory properties of SEE, so it is necessary to know the type of protein–carbohydrate interactions and their effect on these properties. The objective of this research was to study the interaction of proteins and carbohydrates of SEE elaborated with squid mantle, potato and corn. The nutritional composition and protein digestibility were evaluated, Fourier transform infrared (FTIR) and Differential Scanning Calorimetry (DSC) were used to study the formation of protein–starch complexes and the possible regions responsible for their interactions. The SEE had a high protein content (40–85%) and biological value (>93%). The melting temperature (Tm) was found between 145 and 225 °C; the Tm values in extruded samples are directly proportional to the squid content. The extrusion process reduced the amine groups I and II responsible for the protein–protein interaction and increased the O-glucosidic bonds, so these bonds could be responsible for the protein–carbohydrate interactions.     

Red–Green–Blue Trichromophoric Nanoparticles with Dual Fluorescence Resonance Energy Transfer: Highly Sensitive Fluorogenic Response Toward Polyanions

A red–green–blue (RGB) trichromophoric fluorescent organic nanoparticle exhibiting multi‐colour emission was constructed; the blue‐emitting cationic oligofluorene nanoparticle acted as an energy‐donor scaffold to undergo fluorescence resonance energy transfer (FRET) to a red‐emitting dye embedded in the nanoparticle (interior FRET) and to a green‐emitting dye adsorbed on the surface through electrostatic interactions (exterior FRET). Each FRET event occurs independently and is free from sequential FRET, thus the resultant dual‐FRET system exhibits multi‐colour emission, including white, in aqueous solution and film state. A characteristic white‐emissive nanoparticle showed visible responses upon perturbation of the exterior FRET efficiency by acceptor displacement, leading to highly sensitive responses toward polyanions in a ratiometric manner. Specifically, our system exhibits high sensitivity toward heparin with an extremely low detection limit.     

High‐Performance Förster Resonance Energy Transfer (FRET)‐Based Dye‐Sensitized Solar Cells: Rational Design of Quantum Dots for Wide Solar‐Spectrum Utilization

High‐performance Förster resonance energy transfer (FRET)‐based dye‐sensitized solar cells (DSSCs) have been successfully fabricated through the optimized design of a CdSe/CdS quantum‐dot (QD) donor and a dye acceptor. This simple approach enables quantum dots and dyes to simultaneously utilize the wide solar spectrum, thereby resulting in high conversion efficiency over a wide wavelength range. In addition, major parameters that affect the FRET interaction between donor and acceptor have been investigated including the fluorescent emission spectrum of QD, and the content of deposited QDs into the TiO₂ matrix. By judicious control of these parameters, the FRET interaction can be readily optimized for high photovoltaic performance. In addition, the as‐synthesized water‐soluble quantum dots were highly dispersed in a nanoporous TiO₂ matrix, thereby resulting in excellent contact between donors and acceptors. Importantly, high‐performance FRET‐based DSSCs can be prepared without any infrared (IR) dye synthetic procedures. This novel strategy offers great potential for applications of dye‐sensitized solar cells.     

Homogeneous Fluorescence Resonance Energy Transfer Immunoassay for the Determination of Zearalenone

This study demonstrates the use of antigen-antibody binding for the detection of zearalenone. Based on the principle of the fluorescence resonance energy transfer (FRET) phenomenon between antibody and antigen, an immunoassay, in which zearalenone coupled with the anti-zearalenone antibody, was developed, optimized, and applied. Owing to intrinsic fluorescence properties in basic pH conditions with the optimal cationic surfactant, anti-zearalenone and zearalenone played roles as the respective donor and acceptor in the FRET immunoassay. As the concentration of analyte increased, the antigen/antibody emission intensity ratio (I _430 nm/I _350 nm) was enhanced due to larger amounts of zearalenone/anti-zearalenone complexes. This assay, based on the ratio of intensities (I _430 nm/I _350 nm), displayed high specificity and sensitivity with a detection limit of 0.8 ng mL^?1 for zearalenone. The results obtained from analysis of spiked wheat grain samples were found to be in good agreement with those obtained by employing a direct competitive enzyme-linked immunosorbent assay. The label-free, noncompetitive, and homogeneous FRET immunoassay strategy served as a powerful tool for the simple, rapid, and sensitive quantitative determination of zearalenone in food and feed matrices.     

基于聚合物核壳纳米粒子的Hg~(2+)比率型荧光检测传感器

采用一步聚合的方法,制备了以疏水的聚甲基丙烯酸甲酯(PMMA)为核、亲水性的聚电解质支化聚乙烯亚胺(PEI)为壳的纳米粒子分散液.将供体荧光团4-胺基-7-硝基-N-辛基苯并[1,2,5]噁二唑(NBD)以包埋的方式在聚合过程中直接引入PMMA核内部,而受体荧光团罗丹明衍生物SRHB通过吸附作用进入PEI-PMMA核壳界面,构成了含有两种不同荧光分子且可对Hg2+进行荧光比率检测的传感器.考察了含荧光分子的聚合物粒子光谱学性质,证明两种荧光分子均被引入了聚合物粒子体系.在汞离子的荧光检测试验中,加入Hg2+后,体系中的NBD荧光强度下降,而罗丹明的特征发射峰在579 nm处出现,并随着Hg2+浓度的增加,受体/供体的荧光强度比值呈现增长趋势.研究还发现,聚合物粒子基荧光探针对于Hg2+具有较好的选择性,且最佳使用范围是体系pH值在5～8之间,其检测Hg2+的最低浓度可达到1μmol/L.     

FRET-based modified graphene quantum dots for direct trypsin quantification in urine

A versatile nanoprobe was developed for trypsin quantification with fluorescence resonance energy transfer (FRET). Here, fluorescence graphene quantum dot is utilized as a donor while a well-designed coumarin derivative, CMR2, as an acceptor. Moreover, bovine serum albumin (BSA), as a protein model, is not only served as a linker for the FRET pair, but also a fluorescence enhancer of the quantum dots and CMR2. In the presence of trypsin, the FRET system would be destroyed when the BSA is digested by trypsin. Thus, the emission peak of the donor is regenerated and the ratio of emission peak of donor/emission peak of acceptor increased. By the ratiometric measurement of these two emission peaks, trypsin content could be determined. The detection limit of trypsin was found to be 0.7 μg/mL, which is 0.008-fold of the average trypsin level in acute pancreatitis patient's urine suggesting a high potential for fast and low cost clinical screening.     

Probing Deviation of Adhered Membrane Dynamics between Reconstituted Liposome and Cellular System

The dynamics of cell‐cell adhesion are complicated due to complexities in cellular interactions and intra‐membrane interactions. In the present work, we have reconstituted a liposome‐based model system to mimic the cell‐cell adhesion process. Our model liposome system consists of one fluorescein‐tagged and one TRITC (tetramethyl‐rhodamine isothiocyanate)‐tagged liposome, adhered through biotin‐neutravidin interaction. We monitored the adhesion process in liposomes using Förster Resonance Energy Transfer (FRET) between fluorescein (donor) and TRITC (acceptor). Occurrence of FRET is confirmed by the decrease in donor lifetime as well as distinct rise time of the acceptor fluorescence. Interestingly, the acceptor's emission exhibits fluctuations in the range of ≈3±1 s. This may be attributed to structural oscillations associated in two adhered liposomes arising from the flexible nature of biotin‐neutravidin interaction. We have compared the dynamics in a cell‐mimicking liposome system with that in an in vitro live cell system. In the adhered live cell system, we used CPM (7‐diethylamino‐3‐(4‐maleimido‐phenyl)‐4‐methylcoumarin, donor) and nile red (acceptor), which are known to stain the membrane of CHO (Chinese Hamster Ovary) cells. The dynamics of the adhered membranes of two live CHO cells were observed through FRET between CPM and nile red. The acceptor fluorescence intensity exhibits an oscillation in the time‐scale of ≈1±0.75 s, which is faster compared to the reconstituted liposome system, indicating the contributions and involvement of multiple dynamic protein complexes around the cell membrane. This study offers simple reconstituted model systems to understand the complex membrane dynamics using a FRET‐based physical chemistry approach.     

Combining Molecular Dynamic Information and an Aspherical-Atom Data Bank in the Evaluation of the Electrostatic Interaction Energy in Multimeric Protein-Ligand Complex: A Case Study for HIV-1 Protease

Computational analysis of protein–ligand interactions is of crucial importance for drug discovery. Assessment of ligand binding energy allows us to have a glimpse of the potential of a small organic molecule to be a ligand to the binding site of a protein target. Available scoring functions, such as in docking programs, all rely on equations that sum each type of protein–ligand interactions in order to predict the binding affinity. Most of the scoring functions consider electrostatic interactions involving the protein and the ligand. Electrostatic interactions constitute one of the most important part of total interactions between macromolecules. Unlike dispersion forces, they are highly directional and therefore dominate the nature of molecular packing in crystals and in biological complexes and contribute significantly to differences in inhibition strength among related enzyme inhibitors. In this study, complexes of HIV-1 protease with inhibitor molecules (JE-2147 and darunavir) were analyzed by using charge densities from the transferable aspherical-atom University at Buffalo Databank (UBDB). Moreover, we analyzed the electrostatic interaction energy for an ensemble of structures, using molecular dynamic simulations to highlight the main features of electrostatic interactions important for binding affinity.     

A photochromic acceptor as a reversible light-driven switch in fluorescence resonance energy transfer (FRET)

A method has been developed for the quantitative determination of fluorescence resonance energy transfer (FRET) based on the modulation of donor fluorescence upon the reversible photoconversion of a photochromic acceptor. A model system was devised, consisting of Lucifer Yellow cadaverine (LYC, donor) conjugated to the photochromic molecule, 6-nitroBIPS (1′,3′-dihydro-1′-(2-carboxyethyl)-3′,3′-dimethyl-6-nitrospiro[2H-1-benzopyran-2,2′-(2H)-indoline]). Near-ultraviolet irradiation catalyzes the conversion of the colorless spiropyran (SP) to the colored merocyanine (MC) form of 6-nitroBIPS. Only the MC form absorbs at the emission wavelengths of the donor, thereby potentiating FRET, as demonstrated by quenching of the donor. Subsequent irradiation in the visible MC absorption band reverts 6-nitroBIPS to the SP form and FRET is inactivated. The acceptor exhibited high photostability under repeated cycles of alternating UV–Vis irradiation. In this model system, the intramolecular FRET efficiency was close to 100%. The observed maximal donor quenching of 34±3% was indicative of an equilibrium determined by the high quantum efficiency of forward conversion (SP→MC) induced by near-UV irradiation and a low but finite quantum efficiency of the back reaction resulting from excitation of the MC form directly as well as indirectly (by FRET via the donor). A quantitative formalism for the photokinetic scheme was developed. Photochromic FRET (pcFRET) permits repeated, quantitative, and non-destructive FRET determinations for arbitrary relative concentrations of donor and acceptor and thus offers great potential for monitoring dynamic molecular interactions in living cells over extended observation times by fluorescence microscopy.     

Modeling the Dynamics of Protein–Protein Interfaces,How and Why?

Protein–protein assemblies act as a key component in numerous cellular processes. Their accurate modeling at the atomic level remains a challenge for structural biology. To address this challenge, several docking and a handful of deep learning methodologies focus on modeling protein–protein interfaces. Although the outcome of these methods has been assessed using static reference structures, more and more data point to the fact that the interaction stability and specificity is encoded in the dynamics of these interfaces. Therefore, this dynamics information must be taken into account when modeling and assessing protein interactions at the atomistic scale. Expanding on this, our review initially focuses on the recent computational strategies aiming at investigating protein–protein interfaces in a dynamic fashion using enhanced sampling, multi-scale modeling, and experimental data integration. Then, we discuss how interface dynamics report on the function of protein assemblies in globular complexes, in fuzzy complexes containing intrinsically disordered proteins, as well as in active complexes, where chemical reactions take place across the protein–protein interface.     

Quantitative FRET analysis with the EGFP-mCherry fluorescent protein pair

Fluorescence resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful tool to investigate protein–protein interaction and even protein modifications in living cells. Here, we analyze the E⁰GFP-mCherry pair and show that it can yield a reproducible quantitative determination of the energy transfer efficiency both in vivo and in vitro . The photophysics of the two proteins is reported and shows good spectral overlap (Förster radius R ₀ = 51 Å), low crosstalk between acceptor and donor channels, and independence of the emission spectra from pH and halide ion concentration. Acceptor photobleaching (APB) and one- and two-photon fluorescence lifetime imaging microscopy (FLIM) are used to quantitatively determine FRET efficiency values. A FRET standard is introduced based on a tandem construct comprising donor and acceptor together with a 20 amino acid long cleavable peptidic linker. Reference values are obtained via enzymatic cleavage of the linker and are used as benchmarks for APB and FLIM data. E⁰GFP-mCherry shows ideal properties for FLIM detection of FRET and yields high accuracy both in vitro and in vivo . Furthermore, the recently introduced phasor approach to FLIM is shown to yield straightforward and accurate two-photon FRET efficiency data even in suboptimal experimental conditions. The consistence of these results with the reference method (both in vitro and in vivo ) reveals that this new pair can be used for very effective quantitative FRET imaging.     

Phage-Display Based Discovery and Characterization of Peptide Ligands against WDR5

WD40 is a ubiquitous domain presented in at least 361 human proteins and acts as scaffold to form protein complexes. Among them, WDR5 protein is an important mediator in several protein complexes to exert its functions in histone modification and chromatin remodeling. Therefore, it was considered as a promising epigenetic target involving in anti-cancer drug development. In view of the protein–protein interaction nature of WDR5, we initialized a campaign to discover new peptide-mimic inhibitors of WDR5. In current study, we utilized the phage display technique and screened with a disulfide-based cyclic peptide phage library. Five rounds of biopanning were performed and isolated clones were sequenced. By analyzing the sequences, total five peptides were synthesized for binding assay. The four peptides are shown to have the moderate binding affinity. Finally, the detailed binding interactions were revealed by solving a WDR5-peptide cocrystal structure.     

Determination of the Protein-Protein Interactions within Acyl Carrier Protein (MmcB)-Dependent Modifications in the Biosynthesis of Mitomycin

Mitomycin has a unique chemical structure and contains densely assembled functionalities with extraordinary antitumor activity. The previously proposed mitomycin C biosynthetic pathway has caused great attention to decipher the enzymatic mechanisms for assembling the pharmaceutically unprecedented chemical scaffold. Herein, we focused on the determination of acyl carrier protein (ACP)-dependent modification steps and identification of the protein–protein interactions between MmcB (ACP) with the partners in the early-stage biosynthesis of mitomycin C. Based on the initial genetic manipulation consisting of gene disruption and complementation experiments, genes mitE, mmcB, mitB, and mitF were identified as the essential functional genes in the mitomycin C biosynthesis, respectively. Further integration of biochemical analysis elucidated that MitE catalyzed CoA ligation of 3-amino-5-hydroxy-bezonic acid (AHBA), MmcB-tethered AHBA triggered the biosynthesis of mitomycin C, and both MitB and MitF were MmcB-dependent tailoring enzymes involved in the assembly of mitosane. Aiming at understanding the poorly characterized protein–protein interactions, the in vitro pull-down assay was carried out by monitoring MmcB individually with MitB and MitF. The observed results displayed the clear interactions between MmcB and MitB and MitF. The surface plasmon resonance (SPR) biosensor analysis further confirmed the protein–protein interactions of MmcB with MitB and MitF, respectively. Taken together, the current genetic and biochemical analysis will facilitate the investigations of the unusual enzymatic mechanisms for the structurally unique compound assembly and inspire attempts to modify the chemical scaffold of mitomycin family antibiotics.     

Biotin induced fluorescence enhancement in resonance energy transfer and application for bioassay

A novel method to significantly enhance fluorescence resonance energy transfer (FRET) signal which occurred from fluoresceine isothiocyanate (FITC) to Dylight 549 was studied in this paper. Streptavidin was labeled with the donor fluorophore FITC and biotinamide was conjugated to the acceptor Dylight 549. When biotinamide bound to streptavidin, FRET would occur from FITC to Dylight 549 while a remarkable fluorescence enhancement of streptavidin-FITC was observed. The fluorescence enhancement of streptavidin-FITC in the presence of biotin was utilized in the FRET system to obtain higher fluorescence signal. Increase of fluorescence intensity of FITC and decrease of Dylight 549 depended on the concentration of competitive biotin. A homogeneous analysis method was established based on the fluorescence recovery of FITC in the FRET system with fluorescence enhancement. This method is highly sensitive and simple to determine the concentration of biotin. The detection limit for biotin was 0.5 nM and the linear range of the assay was 0.8-9.8 nM. The response time is no more than 15 min during the one-step assay due to the high affinity between streptavidin and biotin.     

Proteomic Studies of Syk-Interacting Proteins Using a Novel Amine-Specific Isotope Tag and GFP Nanotrap

Green fluorescent protein (GFP) and variants have become powerful tools to study protein localization, interactions, and dynamics. We present here a mass spectrometry-based proteomics strategy to examine protein–protein interactions using anti-GFP single-chain antibody V_HH in a combination with a novel stable isotopic labeling reagent, isotope tag on amino groups (iTAG). We demonstrate that the single-chain V_HH (GFP nanotrap) allows us to identify interacting partners of the Syk protein-tyrosine kinase bearing a GFP epitope tag with high efficiency and high specificity. Interacting proteins identified include CrkL, BLNK, α- and β-tubulin, Csk, RanBP5 and DJ-1. The iTAG reagents were prepared with simple procedures and characterized with high accuracy in the determination of peptides in model peptide mixtures and as well as in complex mixture. Applications of the iTAG method and GFP nanotrap to an analysis of the nucleocytoplasmic trafficking of Syk led to the identification of location-specific associations between Syk and multiple proteins. While the results reveal that the new quantitative proteomic strategy is generally applicable to integrate protein interaction data with subcellular localization, extra caution should be taken in evaluating the results obtained by such affinity purification strategies as many interactions appear to occur following cell lysis.     

A FRET-based fluorescent probe for imaging H2S in living cells

A FRET-based fluorescence probe was developed for selective detection of H₂S in aqueous buffer and inside living cells. For this probe, the FRET probe could be cleaved by H₂S, and the fluorescence of FRET donor is released. The probe is highly selective to H₂S over other biologically relevant species to give color change for naked eye observation. Confocal imaging indicated that the probe could monitor intracellular H₂S level changes.