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Int. J. Mol. Sci. 2015, 16(7), 16642-16654; doi:10.3390/ijms160716642

Genetically Encoded FRET-Sensor Based on Terbium Chelate and Red Fluorescent Protein for Detection of Caspase-3 Activity

1
A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, 119071 Moscow, Russia
2
M. V. Lomonosov Moscow State University, Department of Chemistry, 119991 Moscow, Russia
3
Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 142290 Pushchino, Russia
4
Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 142290 Pushchino, Russia
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed.
Academic Editor: Herbert Schneckenburger
Received: 30 April 2015 / Revised: 30 June 2015 / Accepted: 6 July 2015 / Published: 22 July 2015
(This article belongs to the Special Issue Förster Resonance Energy Transfer (FRET) 2015)
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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. View Full-Text
Keywords: FRET-sensor; caspase-3; terbium; fluorescent proteins; apoptosis FRET-sensor; caspase-3; terbium; fluorescent proteins; apoptosis
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Goryashchenko, A.S.; Khrenova, M.G.; Bochkova, A.A.; Ivashina, T.V.; Vinokurov, L.M.; Savitsky, A.P. Genetically Encoded FRET-Sensor Based on Terbium Chelate and Red Fluorescent Protein for Detection of Caspase-3 Activity. Int. J. Mol. Sci. 2015, 16, 16642-16654.

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