Fluorescence energy transfer-sensitized photobleaching of a fluorescent label as a tool to study donor-acceptor distance distributions and dynamics in protein assemblies: studies of a complex of biotinylated IgM with streptavidin and aggregates of concanavalin A.
ABSTRACT A photokinetic method of detection of fluorescence resonance energy transfer (FRET) between special fluorescent labels is applied to study time-averaged spatial distribution of labeled proteins in protein assemblies. Prolonged irradiation of a sample at the absorption maximum of the energy donor label initiates FRET-sensitized fluorescence photobleaching of the energy acceptor label, which was monitored by steady-state fluorimetric measurements. Kinetics of the acceptor photobleaching and kinetics of decreasing the efficiency of FRET from donors to unbleached acceptors were determined. The FRET efficiency was found from measuring sensitization of acceptor fluorescence. Analysis of the photokinetic data permits to estimate the time-averaged distribution of acceptors on donor-acceptor distances in the range of characteristic distances of FRET. Dynamic processes influencing donor-acceptor distances can be also investigated by the method. Application of the method is demonstrated by the studies of a complex of biotinylated IgM with streptavidin and aggregates composed of concanavalin A and sodium dodecyl sulphate. A new thiadicarbocyanine dye was used as the acceptor label, R-phycoerythrin and tetramethylrhodamine isothio-cyanate were the donor labels. In the IgM-streptavidin complex, 16% of acceptors most contributed to FRET provided 90% of FRET efficiency, whereas acceptors made about the same time-averaged contribution to FRET in the concanavalin A aggregates.
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ABSTRACT: Fluorescence resonance energy transfer (FRET) is an extremely effective tool to detect molecular interaction at suboptical resolutions. One of the techniques for measuring FRET is acceptor photobleaching: the increase in donor fluorescence after complete acceptor photobleaching is a measure of the FRET efficiency. However, in wide-field microscopy, complete acceptor photobleaching is difficult due to the low excitation intensities. In addition, the method is sensitive to inadvertent donor bleaching, autofluorescence and bleed-through of excitation light. In the method introduced in this paper, donor and acceptor intensities are monitored continuously during acceptor photobleaching. Subsequently, curve fitting is used to determine the FRET efficiency. The method was demonstrated on cameleon (YC2.1), a FRET-based Ca(2+) indicator, and on a CFP-YFP fusion protein expressed in HeLa cells. FRET efficiency of cameleon in the presence of 1 mm Ca(2+) was 31 +/- 3%. In the absence of Ca(2+) a FRET efficiency of 15 +/- 2% was found. A FRET efficiency of 28% was found for the CFP-YFP fusion protein in HeLa cells. Advantages of the method are that it does not require complete acceptor photobleaching, it includes correction for spectral cross-talk, donor photobleaching and autofluorescence, and is relatively simple to use on a normal wide-field microscope.Journal of Microscopy 07/2005; 218(Pt 3):253-62. DOI:10.1111/j.1365-2818.2005.01483.x · 2.15 Impact Factor
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ABSTRACT: This thesis deals with the development of new techniques and luminescent markers, to improve the quality of luminescence studies in microscopy. A sensitive spectrograph that can be used for spectrally resolved emission spectroscopy in the microscope is described, including design considerations, specifications and applications. The spectrograph is applied to spectral imaging studies of Förster Resonance Energy Transfer (FRET) and the study the photo-physical properties of fluorescence dyes and enhanced mutants of the Green Fluorescent Proteins. Furthermore, the spectrograph is applied to study the luminescence properties of single quantum dots by monitoring the luminescence spectrum of microvolumes with millisecond time-resolution. Part of this thesis describes an analysis method that uses Monte Carlo Simulations to describe the temporal and spatial variations of the fluorescence intensity in a microscopic sample that exhibits FRET, but suffers from photobleaching. Furthermore the Monte Carlo simulations were applied to verify the suitability of a more simple analysis method to calculate the FRET efficiency.
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ABSTRACT: The multiparameter nature of fluorescence microscopy makes it a powerful tool for the investigation of cellular function. Förster resonance energy transfer (FRET) allows the study of functional mechanisms, i.e., protein interactions, modifications, and conformational changes, at the molecular level. Of the different FRET microscopy techniques, fluorescence lifetime imaging (FLIM) provides a quantitative, robust, and sensitive read-out in living cells. The different consequences of the occurrence of FRET and the different forms of FRET bioassays are reviewed here. However, knowledge of isolated biochemical events in cells is not sufficient to understand the working of the highly interconnected cellular pathways. The expansion of the detection principle of FRET could uncover correlations between different components and events. The current focus is on the development of multipoint FRET assays that provide high-detail overviews of functional, structural, and organizational aspects of cellular machine components from which rules and causalities can be derived.01/1970: pages 131-152;