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Combining TIR and FRET in Molecular Test Systems

  • Pharmaceutical agents or drugs often have a 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 measuring the 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. For the TIR-FRET screening of larger cell collectives, we performed three separate 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 to 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.

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Metadaten
Author:Herbert Schneckenburger, Petra Weber, Michael Wagner, Sandra Enderle, Bernd Kalthof, Linn Schneider, Claudia Herzog, Julian Weghuber, Peter Lanzerstorfer
Institutional Author:Herbert Schneckenburger
Institutional Author:Petra Weber
Institutional Author:Michael Wagner
DOI:https://doi.org/10.3390/ijms20030648
ISSN:1661-6596
ISSN:1661-6596
eISSN:1422-0067
Source Title (English):International Journal of Molecular Sciences
Document Type:Article
Language:English
Year of Completion:2019
Release Date:2021/05/11
Volume:20
Number of Pages:11
Faculty:Optik und Mechatronik
Open Access:Open Access
Relevance:nicht peer reviewed