On 22 August 2016 at 15:00 Kadri Ligi will defend her doctoral thesis titled "Characterization and application of protein kinase-responsive organic probes with triplet-singlet energy transfer" in the Faculty of Science and Technology of the University of Tartu in Chemicum (Ravila 14a-1021).
Lead Research Fellow Asko Uri (PhD), Institute of Chemistry, University of Tartu
Research Fellow Erki Enkvist (PhD), Institute of Chemistry, University of Tartu
Professor Niko Hildebrandt, NanoBioPhotonics, Institut d’Electronique Fondamentale, Université Paris-Sud (France)
Enzymes are proteins that catalyze biochemical reactions. Enzymes participate in signal transduction pathways and metabolic pathways and thereby affect the development and growth of cells and how cells react to environmental stimuli. The abnormal three-dimensional structure, localization and activity of an enzyme in cells may lead to diseases. The early detection of changes in the structure, localization profile and activity in cells requires the application of sensitive measurement techniques. The current project was conducted to study one possible approach for the design of sensitive probes for enzymes.
The probes that were applied for the study [ARC-Lum(Fluo) probes] have been developed in the Institute of Chemistry at the University of Tartu. ARC-Lum(Fluo) probes bind to a group of enzymes that are termed basophilic protein kinases (PKs). Upon binding to a PK ARC-Lum(Fluo) probes possess photoluminescence decay time in the microsecond range. The high brightness of the probes is achieved by intramolecular Förster resonant energy transfer (FRET) from a low-QY donor phosphor linked to short-lifetime acceptor with high QY. The long-lifetime donor induces slow acceptor decay, which is due to Förster resonant energy transfer (FRET) from the exited triplet state of the donor (3D*) to the singlet state of the acceptor (1A*). The energy is stored in 3D* and released gradually via FRET to the acceptor fluorescent dye, leading to 1A* of the latter and the following light emission from the dye. The long luminescence lifetime of the probes enables the application of time-gated measurement techniques, which effectively separate the microsecond-scale signal from the background fluorescence of biological samples that have mainly nanosecond-scale lifetime.
The current study was performed to characterize the photoluminescence properties of ARC-Lum(Fluo) probes and to determine the applicability of the probes for mapping and monitoring the activity of PKs in live cells by time-gated luminescence microscopy. The obtained knowledge can be applied for further improvement of organic probes for analysis of different proteins in biological samples as we have shown to be possible for PKs.