title: Genetically Encoded FRET-Sensor Based on Terbium Chelate and Red Fluorescent Protein for Detection of Caspase-3 Activity.
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.
authors.affiliation: A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, 119071 Moscow, Russia. asgoryash@inbi.ras.ru.
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Goryashchenko
authors.affiliation: M. V. Lomonosov Moscow State University, Department of Chemistry, 119991 Moscow, Russia. wasabiko13@gmail.com.
authors.forename: Maria G
authors.initials: MG
authors.lastname: Khrenova
authors.affiliation: M. V. Lomonosov Moscow State University, Department of Chemistry, 119991 Moscow, Russia. bochka567@gmail.com.
authors.forename: Anna A
authors.initials: AA
authors.lastname: Bochkova
authors.affiliation: Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 142290 Pushchino, Russia. ivashina@ibpm.pushchino.ru.
authors.forename: Tatiana V
authors.initials: TV
authors.lastname: Ivashina
authors.affiliation: Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 142290 Pushchino, Russia. levino@bibch.ru.
authors.forename: Leonid M
authors.initials: LM
authors.lastname: Vinokurov
authors.affiliation: A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, 119071 Moscow, Russia. apsavitsky@inbi.ras.ru.|M. V. Lomonosov Moscow State University, Department of Chemistry, 119991 Moscow, Russia. apsavitsky@inbi.ras.ru.
authors.forename: Alexander P
authors.initials: AP
authors.lastname: Savitsky
mesh_terms: D000595:Amino Acid Sequence; D015374:Biosensing Techniques / Q000379:methods*; D053148:Caspase 3 / Q000737:chemistry* / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D008667:Metalloproteins / Q000737:chemistry / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D013725:Terbium / Q000737:chemistry / Q000494:pharmacology*; D000097573:Red Fluorescent Protein
chemical_list: D053148:Caspase 3; D008164:Luminescent Proteins; D008667:Metalloproteins; D010455:Peptides; D013725:Terbium; D000097573:Red Fluorescent Protein
keywords: FRET-sensor; apoptosis; caspase-3; fluorescent proteins; terbium