H. Schneckenburger, P. Weber, M. Wagner, S. Enderle, B. Kalthof, L. Schneider, C. Herzog, J. Weghuber, P. Lanzerstorfer - Combining TIR and FRET in Molecular Test Systems - International Journal of Molecular Sciences, Vol. 1, No. 1, 2019
Pharmaceutical agents or drugs often have some pronounced impact on protein-protein interactions in cells and, in particular, cell membranes. Changes of molecular conformations as well as of intermolecular interactions may affect dipole-dipole interaction between chromophoric groups, which can be proven by measurement of Förster Resonance Energy Transfer (FRET). If these chromophores are located within or in close proximity to the plasma membrane, they are excited preferentially by an evanescent electromagnetic wave upon Total Internal Reflection (TIR) of an incident laser beam. In view of TIR-FRET screening of larger cell collectives we performed three individual steps: (1) setting up of a membrane associated test system for probing the interaction between the Epidermal Growth Factor Receptor (EGFR) and the Growth factor receptor-bound protein 2, (2) use of the Epac-SH188 sensor for quantitative evaluation under the microscope, and (3) application of a TIR fluorescence reader to probe the interaction of GFP with Nile Red. In the first two steps we measured FRET from Cyan (CFP) to Yellow Fluorescent Protein (YFP) by spectral analysis and Fluorescence Lifetime Imaging (FLIM) upon illumination of whole cells (epi-illumination) as well as selective illumination of their plasma membranes by TIR. In particular, TIR excitation permitted FRET measurements with high sensitivity and low background. The Epac sensor showed a more rapid response towards pharmaceutical agents, e.g. Forskolin or the A2B adenosine receptor agonist NECA, in close proximity to the plasma membrane compared to the cytosol. Finally, FRET from a membrane associated GFP to Nile Red was used to test a multi-well TIR fluorescence reader with simultaneous detection of a larger number of samples.