Single-Molecule characterization of FRET-based biosensors and development of Two-Color coincidence detection

Höfig, Henning; Fitter, Jörg (Thesis advisor); Wöll, Dominik (Thesis advisor)

Jülich : Forschungszentrum Jülich GmbH, Zentralbibliothek, Verlag (2020)
Book, Dissertation / PhD Thesis

In: Schriften des Forschungszentrums Jülich. Reihe Schlüsseltechnologien/ key technologies 225
Page(s)/Article-Nr.: 1 Online-Ressource (XVIII, 160 Seiten) : Illustrationen, Diagramme

Dissertation, RWTH Aachen University, 2020

Abstract

Biomolecules often exhibit heterogeneous properties, e. g. they can exist in different conformational states. Ensemble measurements provide only averaged values and, hence, cannot resolve the underlying heterogeneity. In contrast, single-molecule techniques are based on a molecule-by-molecule analysis which allows to reveal the heterogeneity. In this work, two single-molecule methods based on the confocal detection of fluorescent biomolecules are presented. First, genetically-encoded FRET-based biosensors are investigated that utilize Förster resonance energy transfer (FRET) between two fluorescent proteins as the sensor signal. FRET-based biosensors have great potential for applications in biotechnology or diagnostics. Yet, the development of highly-sensitive sensor constructs is complicated by the complexity of the sensor design and the limited information content of the ensemble characterization. This work presents the experimental requirements for the single-molecule detection of FRET-based biosensors followed by a comprehensive single-molecule study of a set of FRET-based biosensors for the determination of glucose concentrations. The single-molecule characterization allows to dissect different parameters that contribute to the sensor performance and, hence, facilitates a more targeted sensor design. Furthermore, the effect of macromolecular crowding on the glucose sensor and two specific crowding sensor constructs is investigated on the single-molecule level. In order to elucidate the role of the fluorescent proteins for the sensor performance, a dye-labeled analogue of the glucose biosensor is investigated on the single-molecule level. The small changes of the FRET signal upon glucose addition in comparison to the biosensor equipped with fluorescent proteins indicate the importance of the relative fluorophore orientation for the FRET signal, that is unique for the employed fluorescent proteins. The second part of this thesis deals with two-color coincidence detection (TCCD) which enables to investigate the binding of two biomolecules that exhibit fluorescence of two different colors. Existing methods underestimate the coincidence due to an imperfect overlap of the confocal volumes of the different colors. The introduction of a brightness gating (BTCCD) for the single-molecule fluorescence intensity facilitates the selection of molecule trajectories that transverse both confocal volumes and, hence, enables quantitative analyses. The capability of the brightness gating is demonstrated by reproducing the coincidence of various, dual-labeled reference samples. After a rigorous testing of the limits of BTCCD, it is applied to FRET-based biosensors to reveal the donor-only and acceptor-only fractions. Finally, BTCCD is used to characterize protein synthesis in a cell-free protein synthesis system and to reveal a previously unrecognized mode of protein translation initiation in bacteria. The ability of BTCCD to provide quantitative results in combination with the versatility of fluorescence assays turns BTCCD into a helpful tool for further studies of the interaction of biomolecules.