Berlin 2012 – wissenschaftliches Programm
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BP: Fachverband Biologische Physik
BP 7: Posters: Proteins
BP 7.15: Poster
Montag, 26. März 2012, 17:30–19:30, Poster A
Determination of the hydrodynamic radius of GFP-tagRFP FRET-Constructs with Fluorescence Correlation Spectroscopy — •Patrick Hätti1, Franz-Josef Schmitt2, Cornelia Junghans2, Marco Vitali2, and Thomas Friedrich2 — 1Institute of Optics and Atomic Physics, Berlin Institute of Technology, Germany — 2Max Volmer Laboratory for Biophysical Chemistry, Berlin Institute of Technology, Germany
Fluorescence correlation spectroscopy (FCS) is suitable to determine the hydrodynamic radius of single molecules and protein chromophors by taking the G(τ) autocorrelation function and calculating the diffusion time. FCS and fluorescence lifetime measurements were done simultaneously with a novel Fluorescence Lifetime Imaging Microscopy setup (FLIM) with an integrated Single-Photon-Avalanche-Diode (SPAD). We determined the hydrodynamic radius of Green Fluorescent Protein to 3.2 nm. A FRET-construct consisting of the GFP-tagRFP fusion protein was observed with a much larger hydrodynamic radius of 12.6 nm, which might be explained by a more complex geometrical structure of the molecule that has more degrees of freedom. GFP and tagRFP are linked via a 5 amino acid-long linker that connects the two single, barrel-shaped fluorescent proteins. In addition to FCS, the setup allows the determination of the fluorescence lifetimes and therefore the calculation of the center-to-center distance of the molecular transition dipoles according to the theory of Förster Resonance Energy Transfer, which is compared with the hydrodynamic radius.