For Forster Resonance Energy Transfer Research Articles

Broad light harvesting under restricted environment: Photophysical understanding leading to enhanced reactive oxygen species generation

Surfactant self-assemblies are of great importance due to their suitable attachment affinity with the hydrophobic organic sensitizers in aqueous media. In this work, the two organic photo-sensitizers protoporphyrin (PPIX) and squaraine (SQ2), absorbing in the visible and near-infrared region of the solar spectrum respectively, have been used simultaneously as dual sensitizers. These two sensitizers behave as donor-acceptor pair for Fo¨rster resonance energy transfer (FRET) in close proximity of restricted micellar environment. This process of dual sensitization on the surface of cationic Cetyltrimethylammonium bromide (CTAB) micelle has been found to generate enhanced reactive oxygen species (ROS) under white light irradiation due to the resonance energy transfer from donor to an acceptor molecule. The choice of the cationic micellar solution has also been justified on the basis of proper sensitizer attachment position with the micelle due to its suitable electrostatic interaction affinity with the sensitizers. The attachment position of the sensitizers with cationic, anionic and nonionic micellar solutions has been confirmed by picosecond resolve fluorescence transients of the sensitizers in solvents of different polarities. Several control studies have been performed with the respective sensitizers alone in presence of white light irradiation and separately in their own absorption regions to confirm the enhanced ROS generation with dual sensitizers under white light irradiation. This positively charged host micelle and generation of ROS can be useful in potential photo remediation application towards selective anionic pollutant. Some preliminary results are also discussed regarding the possibility of anionic pollutant degradation.

Studying α-Synuclein Misfolding through Förster Resonance Energy Transfer

One of the goals of the Petersson laboratory is to develop new methods to study the misfolding of amyloidogenic proteins. Due to their inherent structural heterogeneity, these proteins can be difficult to characterize by traditional structural methods (e.g. crystallography). Fluorescence spectroscopy provides a powerful tool to study protein folding and misfolding in real-time, but its employment requires labeling the protein of interest with two or more fluorophores. We have developed methods to combine multiple orthogonal labeling techniques to produce homogenous double-labeled α-synuclein for Forster resonance energy transfer (FRET) studies. Ultimately, these studies will aid in understanding protein misfolding implicated in neurodegenerative disease.

A Sensitized Emission Based Calibration of FRET Efficiency for Probing the Architecture of Macromolecular Machines

Macromolecular machines participate in almost every cell biological function. These machines can take the form of well-defined protein structures such as the kinetochore, or more loosely organized protein assemblies like the endocytic coat. The protein architecture of these machines—the arrangement of multiple copies of protein subunits at the nanoscale, is necessary for understanding their cell biological function and biophysical mechanism. Defining this architecture in vivo presents a major challenge. High density of protein molecules within macromolecular machines severely limits the effectiveness of super-resolution microscopy. However, this density is ideal for Forster Resonance Energy Transfer (FRET), which can determine the proximity between neighboring molecules. Here, we present a simple FRET quantitation scheme that calibrates a standard epifluorescence microscope for measuring donor–acceptor separations. This calibration can be used to deduce FRET efficiency fluorescence intensity measurements. This method will allow accurate determination of FRET efficiency over a wide range of values and FRET pair number. It will also allow dynamic FRET measurements with high spatiotemporal resolution under cell biological conditions. Although the poor maturation efficiency of genetically encoded fluorescent proteins presents a challenge, we show that its effects can be alleviated. To demonstrate this methodology, we probe the in vivo architecture of the γ-Tubulin Ring. Our technique can be applied to study the architecture and dynamics of a wide range of macromolecular machines.Electronic supplementary materialThe online version of this article (doi:10.1007/s12195-013-0290-y) contains supplementary material, which is available to authorized users.

Ensemble and single particle photophysical properties (two‐photon excitation, anisotropy, FRET, lifetime, spectral conversion) of commercial quantum dots in solution and in live cells

In this work, we characterized streptavidin-conjugated quantum dots (QDs) manufactured by Quantum Dot Corporation. We present data on: (1) two-photon excitation; (2) fluorescence lifetimes; (3) ensemble and single QD emission anisotropy; (4) QDs as donors for Forster resonance energy transfer (FRET); and (5) spectral conversion of QDs exposed to high-intensity illumination. We also demonstrate the utility of QDs for (1) imaging the binding and uptake of biotinylated transferrin on living cells, and (2) resolving by fluorescence lifetime imaging microscopy (FLIM) signals originating from QDs from those of spatially and spectrally overlapping visible fluorescent proteins (VFPs).

Distance Parameters Derived from Time-Resolved Förster Resonance Energy Transfer Measurements and Their Use in Structural Interpretations of Thermodynamic Quantities Associated with Protein–DNA Interactions

Publisher Summary Parameters that define a distance distribution for Forster resonance energy transfer (FRET) donor and acceptor pairs can be extracted from time-resolved FRET measurements. The ultimate interest in extracting such parameters is in determining distances that are relatively static on a nonosecond time scale. In turn, these distances provide information about multiple conformations of macromolecules and about the kinetics and energetics that link such conformations. Not only the mean distance, but parameters from higher moments of the distribution in particular the width— provide the information about the flexibility of macromolecules. These distances and particularly the changes in distances can be determined to high precision allowing FRET to play a key role in providing highly precise distances in solution. This chapter reviews a few essentials of FRET with attention to experimental and computational methods and shows the relationship of distance parameters to macromolecular changes in the field of DNA and DNA–TATA-binding protein (TBP) interactions. These structural changes in turn are intimately linked to various thermodynamic properties. Parameters that characterize a distance distribution P(R) can be obtained from time-resolved FRET measurements. These measurements can involve various combinations of donor-detected FRET and acceptor-detected FRET constrained by steady state emission intensity differences between the donor and that of the donor in the presence of an acceptor. Highly precise average interdye distances R can ultimately lead to precise intra-molecular distances in solution. The width of the P(R) distribution, σ, preferably and more precisely after removal of the tether contributions, yields a measure of conformational equilibria and of conformational dynamics of the macromolecule to which the probes are attached. FRET measurements combined with equilibrium determinations and with rapid-mixing or relaxation kinetics provide structure-energy, entropy profiles of intermediates and transition states along the reaction coordinate.