BiosensorsCopyright: © Fitter
Glucose induced conformational changes within the sensor-protein (gray) can be detected optically by N- and C-terminal attached fluorescent proteins (blue and red).
A successful application of the FRET spectroscopy is given by the analysis of ligand induced conformational changes in so-called sensor proteins. These proteins can be employed to determine the ligand concentration, for example for glucose, calcium, or special amino acids. By this, sensor proteins can be utilized to characterize the chemical composition within the cytosol. Many of these sensor proteins make use of the venus flytrap principle: The ligand induces a bending and a swiveling twist motion about the hinge with an open conformation in the ligand-free state and a close conformation in the ligand-bound state. To measure this conformational change, variants of a cyan fluorescent protein (FP) and a yellow FP (acceptor), are fused to the amino- and carboxyl-termini of the sensor protein, respectively. The design of this kind of sensors is still a challenge and requires a panoply of different methods. Recently, we established a further methodical approach to analyse and optimize the design of the above mentioned genetically encoded FRET sensors by making use of single molecule FRET. In a cooperation with bioengineers (M. Pohl, FZ-Jülich, IGB-1; A.J. Boerma, DWI, RWTH Aachen University) we demonstrated the usability of single-molecule data for elucidating principles of the sensor design.
Höfig H, Cerminara M , Ritter I , Schöne A , Pohl M , Steffen V , Walter J , Vergara Dal Pont I , Katranidis A , Fitter J
Single-Molecule Studies on a FRET Biosensor: Lessons from a Comparison of Fluorescent Protein Equipped versus Dye-Labeled Species.
Molecules, 23(12), 3105 (2018)
Höfig H, Otten J, Steffen V, Pohl M, Boersma AJ, Fitter J
Genetically Encoded Förster Resonance Energy Transfer-Based Biosensors Studied on the Single-Molecule Level.
ACS Sens., 3, 1462-1470, (2018)