Characterization and Application of Protein Kinase-Responsive Organic Probes with Triplet-Singlet Energy Transfer
Date
2016-07-07
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Abstract
Ensüümid on valgud, mis katalüüsivad biokeemilisi reaktsioone. Ensüümid osalevad signaaliülekande- ja ainevahetusradades, mõjutades seeläbi rakkude arengut ja kasvu ning rakkude reaktsioone keskkonnatingimustele. Ensüümide tavapärasest erinev kolmedimensionaalne struktuur, ensüümi lokalisatsioon ja ensüümi aktiivsus rakkudes võib põhjustada organismi haiguslikke seisundeid. Selleks, et muutusi ensüümide struktuuris, paiknemises ja ensüümide aktiivsuses rakkudes saaks detekteerida juba muutuste varajases arengujärgus, on vaja arendada tundlikke mõõtmismeetodeid. Käesolevas töös uuriti süvitsi ühte võimalikku lähenemist ensüümi-tundlike sondide arendamisele.
Uurimistöös kasutati Tartu Ülikooli keemia instituudis välja arendatud fotoluminestsentssonde [ARC-Lum(Fluo) sonde], mille luminestsentsomadused muutuvad oluliselt seondumisel proteiinkinaasidele. Kui proteiinkinaasidele seondunud ARC-Lum(Fluo) sonde ergastada ultravioletse kiirgusega, siis emiteerivad need pikaealist fotoluminestsentskiirgust, mille eluiga jääb mikrosekundilisse suurusjärku. ARC-Lum(Fluo) sondide pikk luminestsentsi eluiga kompleksis proteiinkinaasiga võimaldab kasutada kompleksi detekteerimiseks aegviivitusega mõõtmistehnikaid, mis tagavad pikaealise luminestsentsi selge eristamise bioloogiliste proovide autofluorestsentsist, mille eluiga jääb tavapäraselt nanosekundilisse suurusjärku. Ühtlasi on tagatud mõõtmismeetodi hea signaal-müra suhe ning võimaldatud proteiinkinaaside detektsioon väga väikeses kontsentratsioonis.
Käesolevas töös uuriti ARC-Lum(Fluo) sondide fotoluminestsentsi omadusi. Lisaks rakendati ARC-Lum(Fluo) sonde proteiinkinaaside seireks elusates rakkudes aegviivitusega luminestsentsmikroskoopia abil. Uurimistöö tulemusi saab kasutada sarnasel põhimõttel disainitud sondide arendamiseks teistele ensüümidele.
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 abundance 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 luminescence 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.
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 abundance 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 luminescence 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.
Description
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Keywords
ensüümid, proteiinkinaasid, luminestsents, sondid, enzymes, protein kinases, luminescence, sondes