Abstract
This article describes the genetically encoded caspase-3 FRET-sensor based on the terbium-binding peptide, cleavable linker with caspase-3 recognition site, and red fluorescent protein TagRFP. The engineered construction performs two induction-resonance energy transfer processes: from tryptophan of the terbium-binding peptide to Tb3+ and from sensitized Tb3+ to acceptor—the chromophore of TagRFP. Long-lived terbium-sensitized emission (microseconds), pulse excitation source, and time-resolved detection were utilized to eliminate directly excited TagRFP fluorescence and background cellular autofluorescence, which lasts a fraction of nanosecond, and thus to improve sensitivity of analyses. Furthermore the technique facilitates selective detection of fluorescence, induced by uncleaved acceptor emission. For the first time it was shown that fluorescence resonance energy transfer between sensitized terbium and TagRFP in the engineered construction can be studied via detection of microsecond TagRFP fluorescence intensities. The lifetime and distance distribution between donor and acceptor were calculated using molecular dynamics simulation. Using this data, quantum yield of terbium ions with binding peptide was estimated.
Highlights
Nowadays biosensors based on the Förster resonance energy transfer (FRET) phenomenon are widely used to study the enzymatic activity in living cells [1]
Unlike previously developed lanthanide sensors, in which cryptand-lanthanide complex was a donor, and allophycocyanin or synthetic fluorophores as acceptor [15,16], the Tb3+-TBP-19-TagRFP sensor is genetically encoded and requires only the additional introduction of terbium ions, which on one hand allows for highly standardized samples for in vitro measurements, and on the other hand in the long term makes its use in experiments on living cells possible
FRET efficiency E and energy transfer rate constant kT as well as lifetime of donor in the presence of acceptor τDA were calculated on the fly for each frame of MD trajectory (Figure 2b)
Summary
Nowadays biosensors based on the Förster resonance energy transfer (FRET) phenomenon are widely used to study the enzymatic activity in living cells [1]. Measurements with time delay detect only sensitized fluorescence of the acceptor It solves the problem of simultaneous fluorescence excitation of the donor and acceptor thereby increasing the dynamic range of measurements and the accuracy of the FRET efficiency determination [5]. Unlike previously developed lanthanide sensors, in which cryptand-lanthanide complex was a donor, and allophycocyanin or synthetic fluorophores as acceptor [15,16], the Tb3+-TBP-19-TagRFP sensor is genetically encoded and requires only the additional introduction of terbium ions, which on one hand allows for highly standardized samples for in vitro measurements, and on the other hand in the long term makes its use in experiments on living cells possible
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