Berlin 2012 – wissenschaftliches Programm
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BP: Fachverband Biologische Physik
BP 7: Posters: Proteins
BP 7.18: Poster
Montag, 26. März 2012, 17:30–19:30, Poster A
Combined time-resolved and integrated analysis of FRET efficiency in genetically expressed GFP-tagRFP fusion proteins — •Jörn Weißenborn1, Franz-Josef Schmitt2, Patrick Hätti1, Cornelia Junghans2, Oliver Schöps1, Ulrike Woggon1, and Thomas Friedrich2 — 1Institute for Optics and Atomic Physics, Berlin Institute of Technology, Germany — 2Max Volmer Laboratory for Biophysical Chemistry, Berlin Institute of Technology, Germany
Förster resonance energy transfer (FRET) was investigated in constructs consisting of GFP-tagRFP fusion proteins with varying distance between donor and acceptor. In the short and long construct, GFP and tagRFP are linked via a 5 and 13 amino acid-long linker, respectively, that connects the two single, barrel-shaped fluorescence proteins. Interestingly, a strong donor quenching of the short-linker FRET construct does not lead to a concomitant rise of the acceptor fluorescence (RFP) with the same amplitude. An accurate analysis of 2-dimensional photoluminescence excitation spectroscopy (PLE) and time-and wavelength resolved fluorescence decay showed congruent results. The data analysis reveals that only 50 % of the GFP molecules are coupled to tagRFP via excitation energy transfer (EET). The efficiency of the EET in the coupled GFP-tagRFP pairs calculates to about 65 %. According to our experiments and the FRET theory, the long-linker construct should show virtually no EET. The overall transfer efficiency (coupled and uncoupled species) is calculated to 30 % and < 1 % in the short- and long-linker FRET construct, respectively.