Berlin 2018 – scientific programme
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BP: Fachverband Biologische Physik
BP 9: Postersession I
BP 9.2: Poster
Monday, March 12, 2018, 17:30–19:30, Poster A
Performance of genetically encoded FRET-based biosensors investigated on single molecule level — •Henning Höfig1,2, Martina Pohl3, Julia Otten3, Arnold Boersma4, and Jörg Fitter1,2 — 1RWTH Aachen University, I. Physikalisches Institut (IA), AG Biophysik, Aachen, Germany — 2Research Centre Juelich, ICS-5, Juelich, Germany — 3Research Centre Juelich, IGB-1, Juelich, Germany — 4University of Groningen, Department of Biochemistry, Groningen, Netherlands
Genetically encoded FRET-based biosensors consist of two fluorescent proteins (donor and acceptor) and a sensing domain. The readout of FRET-based biosensors usually utilizes the ratio of fluorescence emission intensities of the donor and the acceptor upon donor excitation. We carried out single-molecule measurements on a confocal microscope for two types of CFP-YFP biosensors, one sensitive to glucose concentration [1] and another one monitors macromolecular crowding [2]. From our measurements we obtained FRET efficiencies histograms dissecting the different subpopulations of the sensor under varying environmental conditions. In order to demonstrate the capability of utilizing transfer efficiency histograms for judging the performance of FRET-based sensor constructs we analyze various glucose sensor constructs. The obtained smFRET histograms display specific fingerprints of the respective sensor properties and provide a valuable basis for a rational design of FRET-based biosensors.
[1] R. Moussa et al., J. Biotechnol., 191, 250-259 (2014); [2] A. J. Boersma et al., Nat. Methods, 12, 227-229 (2015)