Fluorimetric determination of febuxostat in dosage forms and in real human plasma via Förster resonance energy transfer

A rapid, simple, selective and precise fluorimetric method was developed and validated for determination of a selective xanthine oxidase inhibitor; febuxostat (FBX) in pharmaceutical formulations and in human plasma. The proposed method is based on quenching effect of FBX on the fluorescence intensity of terbium (Tb³⁺) through fluorescence resonance energy transfer (FRET) from Tb³⁺ to FBX. The formed complex was measured at λ_ex. 320 nm/λ_em. 490 nm against a reagent blank. Fluorescence intensity of Tb³⁺ was diminished when FBX was added. A linear relationship between the fluorescence quenching value of the formed complex and the concentration of FBX was investigated. The reaction conditions and the fluorescence spectral properties of the complex have been studied. The linearity range of the developed method was 1.0–16.0 μg/ml. The suggested method was applied successfully for the estimation of FBX in bulk powder, dosage forms and spiked plasma samples with excellent recoveries (96.79–98.89%). In addition, the developed method has been successfully applied for determination of FBX in real plasma samples collected from healthy volunteers with good recoveries (82.06–85.65%). All obtained results of the developed method were statistically analyzed and validated according to ICH (International Conference on Harmonization) guidelines.     

Förster resonance energy transfer measurements of cofactor‐dependent effects on protein arginine N‐methyltransferase homodimerization

Protein arginine N‐methyltransferase (PRMT) dimerization is required for methyl group transfer from the cofactor S‐adenosyl‐L ‐methionine (AdoMet) to arginine residues in protein substrates, forming S‐adenosyl‐L ‐homocysteine (AdoHcy) and methylarginine residues. In this study, we use Förster resonance energy transfer (FRET) to determine dissociation constant (K_D) values for dimerization of PRMT1 and PRMT6. By attaching monomeric Cerulean and Citrine fluorescent proteins to their N‐termini, fluorescent PRMTs are formed that exhibit similar enzyme kinetics to unconjugated PRMTs. These fluorescent proteins are used in FRET‐based binding studies in a multi‐well format. In the presence of AdoMet, fluorescent PRMT1 and PRMT6 exhibit 4‐ and 6‐fold lower dimerization K_D values, respectively, than in the presence of AdoHcy, suggesting that AdoMet promotes PRMT homodimerization in contrast to AdoHcy. We also find that the dimerization K_D values for PRMT1 in the presence of AdoMet or AdoHcy are, respectively, 6‐ and 10‐fold lower than the corresponding values for PRMT6. Considering that the affinity of PRMT6 for AdoHcy is 10‐fold higher than for AdoMet, PRMT6 function may be subject to cofactor‐dependent regulation in cells where the methylation potential (i.e., ratio of AdoMet to AdoHcy) is low. Since PRMT1 affinity for AdoMet and AdoHcy is similar, however, a low methylation potential may not affect PRMT1 function.     

Visualization of human immunodeficiency virus protease inhibition using a novel Förster resonance energy transfer molecular probe

The in vivo high‐throughput screening (HTS) of human immunodeficiency virus (HIV) protease inhibitors is a significant challenge because of the lack of reliable assays that allow the visualization of HIV targets within living cells. In this study, we developed a new molecular probe that utilizes the principles of Förster resonance energy transfer (FRET) to visualize HIV‐1 protease inhibition within living cells. The probe is constructed by linking two fluorescent proteins: AcGFP1 (a mutant green fluorescent protein) and mCherry (a red fluorescent protein) with an HIV‐1 protease cleavable p2/p7 peptide. The cleavage of the linker peptide by HIV‐1 protease leads to separation of AcGFP1 from mCherry, quenching FRET between AcGFP1 and mCherry. Conversely, the addition of a protease inhibitor prevents the cleavage of the linker peptide by the protease, allowing FRET from AcGFP1 to mCherry. Thus, HIV‐1 protease inhibition can be determined by measuring the FRET signal's change generated from the probe. Both in vitro and in vivo studies demonstrated the feasibility of applying the probe for quantitative analyses of HIV‐1 protease inhibition. By cotransfecting HIV‐1 protease and the probe expression plasmids into 293T cells, we showed that the inhibition of HIV‐1 protease by inhibitors can be visualized or quantitatively determined within living cells through ratiometric FRET microscopy imaging measurement. It is expected that this new probe will allow high‐content screening (HCS) of new anti‐HIV drugs. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Quantitative time domain analysis of lifetime‐based Förster resonant energy transfer measurements with fluorescent proteins: Static random isotropic fluorophore orientation distributions

Förster resonant energy transfer (FRET) measurements are widely used to obtain information about molecular interactions and conformations through the dependence of FRET efficiency on the proximity of donor and acceptor fluorophores. Fluorescence lifetime measurements can provide quantitative analysis of FRET efficiency and interacting population fraction. Many FRET experiments exploit the highly specific labelling of genetically expressed fluorescent proteins, applicable in live cells and organisms. Unfortunately, the typical assumption of fast randomization of fluorophore orientations in the analysis of fluorescence lifetime‐based FRET readouts is not valid for fluorescent proteins due to their slow rotational mobility compared to their upper state lifetime. Here, previous analysis of effectively static isotropic distributions of fluorophore dipoles on FRET measurements is incorporated into new software for fitting donor emission decay profiles. Calculated FRET parameters, including molar population fractions, are compared for the analysis of simulated and experimental FRET data under the assumption of static and dynamic fluorophores and the intermediate regimes between fully dynamic and static fluorophores, and mixtures within FRET pairs, is explored. Finally, a method to correct the artefact resulting from fitting the emission from static FRET pairs with isotropic angular distributions to the (incorrect) typically assumed dynamic FRET decay model is presented.      

Native and in vitro-associated phycobilisomes of Nostoc sp. Composition,energy transfer,and effect of antibodies

The relationship of the structure and function of the light-harvesting antennae in the blue-green alga Nostoc sp. was further elucidated by reconstitution experiments. Separated phycoerythrin-phycocyanin complexes and allophycocyanin fractions were reassociated as described earlier (Canaani, O., Lipschultz, C.A. and Gantt, E. (1980) FEBS Lett. 115, 225–229) into functional phycobilisomes with a 70% yield. Native and reassociated physobilisomes had molar ratios of about 1.4:1.1:1.0 of phycoerythrin:phycocyanin:allophycocyanim. Energy transfer was demonstrated by their fluorescence emission maximum at approx. 675 nm (20°C), and their excitation spectra (emission wavelength 680 nm) which reflected the contribution of the three constitutive phycobiliproteins. Scans of Coomassie blue-stained SDS-polyacrylamide gels showed that the polypeptide composition of native and reassociated phycobilisomes was virtually indistinguishable. Reassociation of phycobilisomes was dependent on the interaction of allophycocyanin and phycocyanin, because it could be blocked with antisera to phycocyanin and allophycocyanin, but not to phycoerythrin. In addition, reassociation did not occur when a 31 000 Da polypeptide, which is part of the phycoerythrin-phycocyanin complex, was reduced in size (by 4000 Da). These results suggest that at least two domains are required for functional reassociation of phycobilisomes involving phycocyanin and allophycocyanin.     

A comparison of Förster resonance energy transfer analysis approaches for Nanodrop fluorometry

Ensemble Förster resonance energy transfer (FRET) results can be analyzed in a variety of ways. Due to experimental artifacts, the results obtained from different analysis approaches are not always the same. To determine the optimal analysis approach to use for Nanodrop fluorometry, we have performed both ensemble and single-molecule FRET studies on oligomers of double-stranded DNA. We compared the single-molecule FRET results with those obtained using various ensemble FRET analysis approaches. This comparison shows that for Nanodrop fluorometry, analyzing the increase of the acceptor fluorescence is less likely to introduce errors in estimates of FRET efficiencies compared with analyzing the fluorescence intensity of the donor in the absence and presence of the acceptor.     

Automated fluorescence lifetime imaging plate reader and its application to Förster resonant energy transfer readout of Gag protein aggregation

Fluorescence lifetime measurements can provide quantitative readouts of local fluorophore environment and can be applied to biomolecular interactions via Förster resonant energy transfer (FRET). Fluorescence lifetime imaging (FLIM) can therefore provide a high content analysis (HCA) modality to map protein‐protein interactions (PPIs) with applications in drug discovery, systems biology and basic research. We present here an automated multiwell plate reader able to perform rapid unsupervised optically sectioned FLIM of fixed and live biological samples and illustrate its potential to assay PPIs through application to Gag protein aggregation during the HIV life cycle. We demonstrate both hetero‐FRET and homo‐FRET readouts of protein aggregation and report the first quantitative evaluation of a FLIM HCA assay by generating dose response curves through addition of an inhibitor of Gag myristoylation. Z ′ factors exceeding 0.6 are realised for this FLIM FRET assay. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Visualization of excitation energy transfer processes in plants and algae

Development of the time-resolved fluorescence spectroscopy in the pico-second time range and its application to the energy transfer processes in many photosynthetic organisms is reviewed here. This method enabled visualization of energy transfer processes by three-dimensional expression of fluorescence spectra and discrimination of kinetic components and spectral components. The second generation of the ultrafast fluorescence spectroscopy is the femto-second (fs) fluorescence up-conversion, and this has enabled analyses of the transfer processes from carotenoids to chlorophylls with a resolution of less than 100 fs. For future progress, a further development of the spectroscopy is indispensable as well as structural data at atomic resolution. This revised version was published online in June 2006 with corrections to the Cover Date.     

Chromatin nanoscale compaction in live cells visualized by acceptor‐to‐donor ratio corrected Förster resonance energy transfer between DNA dyes

@Chromatin nanoscale architecture in live cells can be studied by Förster resonance energy transfer (FRET) between fluorescently labeled chromatin components, such as histones. A higher degree of nanoscale compaction is detected as a higher FRET level, since this corresponds to a higher degree of proximity between donor and acceptor molecules. However, in such a system, the stoichiometry of the donors and acceptors engaged in the FRET process is not well defined and, in principle, FRET variations could be caused by variations in the acceptor‐to‐donor ratio rather than distance. Here, to get a FRET level independent of the acceptor‐to‐donor ratio, we combine fluorescence lifetime imaging detection of FRET with a normalization of the FRET level to a pixel‐wise estimation of the acceptor‐to‐donor ratio. We use this method to study FRET between two DNA binding dyes staining the nuclei of live cells. We show that this acceptor‐to‐donor ratio corrected FRET imaging reveals variations of nanoscale compaction in different chromatin environments. As an application, we monitor the rearrangement of chromatin in response to laser‐induced microirradiation and reveal that DNA is rapidly decompacted, at the nanoscale, in response to DNA damage induction.      

A Förster resonance energy transfer sensor for live‐cell imaging of mitogen‐activated protein kinase activity in Arabidopsis

The catalytic activity of mitogen‐activated protein kinases (MAPKs) is dynamically modified in plants. Since MAPKs have been shown to play important roles in a wide range of signaling pathways, the ability to monitor MAPK activity in living plant cells would be valuable. Here, we report the development of a genetically encoded MAPK activity sensor for use in Arabidopsis thaliana. The sensor is composed of yellow and blue fluorescent proteins, a phosphopeptide binding domain, a MAPK substrate domain and a flexible linker. Using in vitro testing, we demonstrated that phosphorylation causes an increase in the Förster resonance energy transfer (FRET) efficiency of the sensor. The FRET efficiency can therefore serve as a readout of kinase activity. We also produced transgenic Arabidopsis lines expressing this sensor of MAPK activity (SOMA) and performed live‐cell imaging experiments using detached cotyledons. Treatment with NaCl, the synthetic flagellin peptide flg22 and chitin all led to rapid gains in FRET efficiency. Control lines expressing a version of SOMA in which the phosphosite was mutated to an alanine did not show any substantial changes in FRET. We also expressed the sensor in a conditional loss‐of‐function double‐mutant line for the Arabidopsis MAPK genes MPK3 and MPK6. These experiments demonstrated that MPK3/6 are necessary for the NaCl‐induced FRET gain of the sensor, while other MAPKs are probably contributing to the chitin and flg22‐induced increases in FRET. Taken together, our results suggest that SOMA is able to dynamically report MAPK activity in living plant cells.     

Structure,assembly and energy transfer of plant photosystem II supercomplex

Around photosystem II (PSII), the peripheral antenna system absorbs sunlight energy and transfers it to the core complex where the water-splitting and oxygen-evolving reaction takes place. The peripheral antennae in plants are composed of various light-harvesting complexes II (LHCII). Recently, the three-dimensional structure of the C₂S₂M₂-type PSII-LHCII supercomplex from Pisum sativum (PsPSII) has been solved at 2.7-Å resolution using the single-particle cryo-electron microscopy method. The large homodimeric supercomplex has a total molecular weight of >1400?kDa. Each monomer has a core complex surrounded by strongly and moderately bound LHCII trimers, as well as CP29, CP26, and CP24 monomers. Here, we review and present a detailed analysis of the structural features of this supramolecular machinery. Specifically, we discuss the structural differences around the oxygen-evolving center of PSII from different species. Furthermore, we summarize the existing knowledge of the structures and locations of peripheral antenna complexes, and dissect the excitation energy transfer pathways from the peripheral antennae to the core complex. This detailed high-resolution structural information provides a solid basis for understanding the functional behavior of plant PSII-LHCII supercomplex.     

Multiway data analysis approach toward understanding of photoluminescence and energy transfer in carbon nanodots

In this study, dilution analysis and anion exchange chromatography (AEC) were employed to provide insights into the photoluminescence (PL) of carbon nanodots (CNDs). A stepwise dilution process revealed that some of the fluorophores with higher energy emission were quenched in the high concentration solution and appeared in the dilute solutions. AEC fractionation led to seven sorts of CND fractions with similar surface charges. The fractionation for this CND mixture showed that excitation wavelength dependence was lower for separated CND particles. The wavelength dependence of excitation spectra could be due to energy exchange between particles that was reduced in diluted solutions and separated fractions. Multivariate analysis of AEC's data demonstrated that there were five distinct fluorophores, which formed the total CND emission. It is interesting that none of these fluorophores had a clear contribution to the surface charge of the CND particles. Further characterization through FTIR spectroscopy and ¹H NMR revealed that optical properties of CNDs did not follow the surface functional groups in CNDs. This situation means that the optical behaviour of particles and their fluorophores differed depending on the surface functional groups.     

Iron deficiency interrupts energy transfer from a disconnected part of the antenna to the rest of Photosystem II

Iron deficiency changed markedly the shape of the leaf chlorophyll fluorescence induction kinetics during a dark-light transition, the so-called Kautsky effect. Changes in chlorophyll fluorescence lifetime and yield were observed, increasing largely the minimal and the intermediate chlorophyll fluorescence levels, with a marked dip between the intermediate and the maximum levels and loss of the secondary peak after the maximum. During the slow changes, the lifetime-yield relationship was found to be linear and curvilinear (towards positive lifetime values) in control and Fe-deficient leaves, respectively. These results suggested that part of the Photosystem II antenna in Fe-deficient leaves emits fluorescence with a long lifetime. In dark-adapted Fe-deficient leaves, measurements in the picosecond-nanosecond time domain confirmed the presence of a 3.3-ns component, contributing to 15% of the total fluorescence. Computer simulations revealed that upon illumination such contribution is also present and remains constant, indicating that energy transfer is partially interrupted in Fe-deficient leaves. Photosystem II-enriched membrane fractions containing different pigment-protein complexes were isolated from control and Fe-deficient leaves and characterized spectrophotometrically. The photosynthetic pigment composition of the fractions was also determined. Data revealed the presence of a novel pigment-protein complex induced by Fe deficiency and an enrichment of internal relative to peripheral antenna complexes. The data suggest a partial disconnection between internal Photosystem II antenna complexes and the reaction center, which could lead to an underestimation of the Photosystem II efficiency in dark-adapted, low chlorophyll Fe-deficient leaves, using chlorophyll fluorescence. This revised version was published online in August 2006 with corrections to the Cover Date.     

The co-translational folding and interactions of nascent protein chains: a new approach using fluorescence resonance energy transfer

During protein biosynthesis, a nascent protein is exposed to multiple environments and proteins both inside and outside the ribosome that influence nascent chain folding and trafficking. Fluorescence resonance energy transfer between two dyes incorporated into a single nascent chain using aminoacyl-tRNA analogs can directly and selectively monitor changes in nascent chain conformation. This approach recently revealed the existence and functional ramifications of ribosome-mediated folding of nascent membrane proteins inside the ribosome and can be extended to characterize the effects of chaperones and other proteins and ligands on nascent protein folding, interactions, assembly, and avoidance of misfolding and degradation.     

Photoinduced electron transfer and energy transfer reactions of hydroxo-(2,2′:6′,2″-terpyridine) platinum(II)

The luminescent complex [Pt(terpy)OH]BF₄ undergoes photoinduced electron transfer reactions with phenyl amine electron donors and nitrophenyl electron acceptors. Stern-Volmer analysis of the quenching of metal-to-ligand charge transfer phosphorescence (³MLCT) was used to calculate bimolecular rate constants for electron transfer. Rate constants vary from 10⁸ to >10¹⁰ M⁻¹ s⁻¹, depending on the thermodynamic driving force of the electron transfer reaction, with rate constants indicating that [Pt(terpy)OH]BF₄* is a powerful photo-oxidant. Aromatic triplet energy acceptors can also quench the ³MLCT emission.     

A molecular dynamics model of the Bt toxin Cyt1A and its validation by resonance energy transfer

Cyt1A is a cytolytic toxin from Bacillus thuringiensis var. israelensis. A computer model of the toxin in solution was generated and validated by resonance energy transfer (RET). The average distance between the two tryptophans (residues 158 and 161) and the fluorescently labeled cysteine 190 was 2.16 nm, which closely matched the distance predicted in computer simulations, 2.2 nm. The simulation results were able to explain two previous experimental observations: (i) amino-acid sequences of all Cyt toxins contain four blocks of highly conserved residues; and (ii) several single-point mutations drastically abrogated Cyt1A's toxicity. Selective randomization of atomic coordinates in the computer model revealed that the conserved blocks are important for proper folding and stability of the toxin molecule. Replacing lysine 225 with alanine, a mutation that renders the toxin inactive, was shown to result in breaking the hydrogen bonds between K225 and V126, L123, and Y189. Calculated Helmholtz free energy difference of the inactive mutation K225A was higher by 12 kcal/mol and 5 kcal/mol than the values for the benign mutations K118A and K198A, respectively, which indicates that the K225A mutant is significantly destabilized. The normal-mode and principal-component analyses revealed that in the wild-type Cyt1A the region around the residue K225 is quite stationary, due to the hydrogen-bond network around K225. In contrast, pronounced twisting and stretching were observed in the mutant K225A, and the region around the residue K225 becomes unstable. Our results indicate that conformational differences in this mutant spread far away from the site of the mutation, suggesting that the mutant is inactivated due to an overall change in conformation and diminished stability rather than due to a localized alteration of a “binding” or “active” site.     

Fluorescence resonance energy transfer between two quantum dots with immunocomplexes of antigen and antibody as a bridge.

In this study, 573 nm quantum dots (QDs)-rabbit IgG-goat anti-rabbit IgG-638 nm QDs immunocomplexes were prepared, utilizing antigen-antibody interaction. 573 nm-emitting QDs were conjugated to antigen (rabbit IgG) and 638 nm-emitting QDs were conjugated to antibody (goat anti-rabbit IgG) via electrostatic/hydrophilic self-assembly, respectively. The mutual affinity of the antigen and antibody brought two kinds of QDs close enough to result in fluorescence resonance energy transfer (FRET) between them; the luminescence emission of 573 nm QDs was quenched, while that of 638 nm QDs was enhanced. The luminescence emission of 573 nm QDs could be recovered when the immunocomplexes were exposed to the unlabelled rabbit IgG antigen. The FRET efficiency (E) and the distance between the donor and the acceptor were calculated.