A FRET approach for luminescence sensing Cr in aqueous solution and living cells through functionalizing glutathione and glucose moieties

A simple FRET-based approach to ratiometric fluorescence sensing of Cr³⁺ in aqueous solution using glutathione and glucose as building blocks was achieved, inspired to the binding motifs of Cr³⁺ in glucose tolerance factors (GTF). Selectivity and competition experiments showed that this system featured high sensitivity (detection limit ≤0.1 ppm) and excellent selectivity over other metal ions in buffer solution. Confocal fluorescence microscopy experiments had established the utility of the approach in monitoring Cr³⁺ within living cells.     

Practical Use of Corrected Fluorescence Excitation and Emission Spectra of Fluorescent Proteins in Förster Resonance Energy Transfer (FRET) Studies

Corrected fluorescence excitation and emission spectra have been obtained from several enhanced variants of the green fluorescent protein (EGFP) isolated from the jellyfish Aequorea victoria, blue fluorescence protein (EBFP), cyan fluorescent protein (ECFP), EGFP and yellow fluorescent protein (EYFP–citrine) and from the red fluorescent protein (DsRed) isolated from the coral species Discosoma. The spectra are stored in a database. This report describes how the spectra can be used as templates to derive the critical transfer distance for any pair of fluorescent proteins.     

Preparation of Fluorescence Tunable Polymer Nanoparticles by One-step Mini-emulsion

Fluorescence tunable polymer nanoparticles were prepared by incorporating two hydrophobic fluorescent dyes (9, 10-diphenylanthracene: DPA and nitrobenzoxadiazolyl: NBD) into polymethylmethacrylate (PMMA) nanoparticles via one-step mini-emulsion polymerization method. The prepared fluorescent nanoparticles exhibit the spectral properties of both DPA and NBD dye, indicating that the two fluorophores have been incorporated into the nanoparticles. The nanoparticles greatly enhance the fluorescence emission of the two hydrophobic dyes in aqueous media probably by providing good protection of the dye molecules in the polymer nanoparticles matrix. Moreover, by varying the doping ratio of the two hydrophobic dyes, the polymer nanoparticles exhibit tunable and distinguishable emission characteristics under a single wavelength excitation via occuring fluorescence resonance energy transfer (FRET).     

Ultrafast dynamics in a nanocage of enzymes: solvation and fluorescence resonance energy transfer in reverse micelles

In this contribution we report studies of the nature of solvation and resonance energy transfer processes in a reverse micelle (RM) upon encapsulation of a digestive enzyme, alpha-chymotrypsin (CHT). We have used one donor, Coumarin 500 (C500), and three acceptors Rhodamine 123 (R123, cationic), ethidium bromide (EtBr, cationic), and Merocyanine 540 (MC540, anionic). By selectively exciting the donor at the surface of the RM with a proper excitation wavelength we have examined solvation dynamics in the microenvironment. The solvation correlation function in the RM without CHT exhibits single-exponential decay with time constant approximately 660 ps, which is similar to that of the CHT-included RM. However, in the case of CHT-included RM (w(0)=10), the time-resolved anisotropy and spectral linewidth analysis of the surface-bound donor reveal the existence of an annular aqueous channel of thickness approximately 2.5 A between the enzyme surface and the inner surface of the RM. The aqueous channel is a potential host for the water-soluble substrate and also is involved in maintaining the proper functionality of RM encapsulated CHT. The studies use both steady-state and time-resolved fluorescence resonance energy transfer (FRET) techniques to measure donor-acceptor distances in the RM and also emphasize the danger of using steady-state fluorescence quenching as a method in careful estimation of the distances. The local geometrical restriction on the donor and acceptor molecules was estimated from time-resolved polarization (anisotropy) measurements. The time-resolved anisotropy of the donor and acceptor molecules also revealed significant randomization of the relative orientation of transition dipoles of the donor and acceptor, justifying the use of 2/3 as the value of the orientation factor kappa2. These studies attempt to elucidate the excellence of the RM as a nanohost of biological macromolecules.     

A dyadic sensitizer for dye solar cells with high energy-transfer efficiency in the device.

A new bichromophoric dyad based on an alkyl-functionalized aminonaphthalimide as energy-donor chromophore and [Ru(dcbpy)2(acac)]Cl (dcbpy=4,4'-dicarboxybipyridine, acac=acetylacetonato) as energy acceptor and sensitizing chromophore is synthesized. Efficient quenching of the donor-chromophore emission is observed in solution, presumably due to resonant energy transfer. This dyad is then used as a sensitizer in a dye solar cell. By comparing the spectral properties of transparent dye solar cells sensitized with the dyad and [Ru(dcbpy)2(acac)]Cl, it is possible to demonstrate that photons absorbed by the donor moiety also contribute significantly to the generation of current. Instead of using acceptor luminescence as a probe, enhanced photocurrent generation is employed to estimate the energy-transfer efficiency. Fitting theoretical to experimental external quantum efficiency functions gives a value for the energy-transfer efficiency of 85 %. Evaluation of the maximum output power of dye solar cells sensitized with the dyad and [Ru(dcbpy)2(acac)]Cl showed, under selective illumination at the absorption maximum of the donor chromophore, that the introduction of the energy-donor moiety leads to a significant increase in the monochromatic maximum output power under blue illumination. This result demonstrates the usefulness of energy transfer for the generation of current in dye-sensitized solar cells.     

Ultrafast fluorescence depolarisation in the yellow fluorescent protein due to its dimerisation.

Transient absorption spectroscopy with sub-100 fs time resolution was performed to investigate the oligomerisation behaviour of eYFP in solution. A single time constant tau(AD)=2.2+/-0.15 ps is sufficient to describe the time-resolved anisotropy decay up to at least 200 ps. The close contact of two protein barrels is deduced as the exclusive aggregation state in solution. From the final anisotropy r(infinity)=0.28+/-0.02, the underlying quaternary structure can be traced back to the somewhat distorted structure of the dimers of wt-GFP. The use of autofluorescent proteins as rulers in F?rster resonance energy transfer (FRET) measurements may demand polarisation-sensitive detection of the fluorescence with high time resolution.     

Biophysical approaches to G protein-coupled receptors: Structure, function and dynamics

G protein-coupled receptors (GPCR) represent a large family of drug targets for which there is no high-resolution structural information. In order to understand the mechanisms of ligand recognition and receptor activation, there is a strong need for novel biophysical methods. In this Perspective we provide an overview of recent experimental approaches used to explore the molecular architecture and dynamics of GPCR and their interactions with ligands and G proteins using biophysical, non-crystallographic, methods.     

Single-molecule fluorescence spectroscopy of protein folding.

Single-molecule spectroscopy is an important new approach for studying the intrinsically heterogeneous process of protein folding. This Review illustrates how different single-molecule fluorescence techniques have improved our understanding of mechanistic aspects in protein folding, exemplified by a series of recent experiments on a small protein.     

Application of a Highly Robust and Efficient Fluorescence‐Resonance‐Energy‐Transfer (FRET) System in DNA

We report a feasibility study for the application of our newly developed highly efficient and robust fluorescence‐resonance‐energy‐transfer (FRET) system to DNA. A 2′‐oligodeoxynucleotide, 12 , equipped with a quinolinone derivative as donor and a (bathophenanthroline)ruthenium(II) complex as acceptor and having a single uridine as potential cleavage site under basic conditions revealed an intensive FRET, which almost vanished after cleavage of the oligonucleotide under basic conditions (Fig. 7). Furthermore, in the arrangement of a molecular beacon (MB) DNA (see 13 ), a significant decrease of the FRET was observed after hybridization to a target sequence (Fig. 9). Due to the long decay times of the fluorescence of the Ru‐complex, the system allows for highly sensitive time‐gated measurements.