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Int J Mol Sci. 2015 Jul 22;16(7):16642-54. doi: 10.3390/ijms160716642.

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

Author information

1
A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, 119071 Moscow, Russia. asgoryash@inbi.ras.ru.
2
M. V. Lomonosov Moscow State University, Department of Chemistry, 119991 Moscow, Russia. wasabiko13@gmail.com.
3
M. V. Lomonosov Moscow State University, Department of Chemistry, 119991 Moscow, Russia. bochka567@gmail.com.
4
Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 142290 Pushchino, Russia. ivashina@ibpm.pushchino.ru.
5
Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 142290 Pushchino, Russia. levino@bibch.ru.
6
A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, 119071 Moscow, Russia. apsavitsky@inbi.ras.ru.
7
M. V. Lomonosov Moscow State University, Department of Chemistry, 119991 Moscow, Russia. apsavitsky@inbi.ras.ru.

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 Tb(3+) and from sensitized Tb(3+) 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.

KEYWORDS:

FRET-sensor; apoptosis; caspase-3; fluorescent proteins; terbium

PMID:
26204836
PMCID:
PMC4519970
DOI:
10.3390/ijms160716642
[Indexed for MEDLINE]
Free PMC Article

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