Induction-resonance energy transfer between the terbium-binding peptide and the red fluorescent proteins DsRed2 and TagRFP

Arslanbaeva, L. R.; Zherdeva, Victoria V.; Ивашина, Т. В.; Vinokurov, Leonid M.; Morozov, V.B.; Olenin, A. N.; Савицкий, А. П.

doi:10.1134/s0006350911030043

Cited by 4 publications

(12 citation statements)

References 24 publications

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“…Registration of spectra was performed with a time delay of 100 μs, allowing to exclude the contribution of the direct excitation of the acceptor to the total intensity, and to measure only the fluorescence of the acceptor due to the energy transfer. In addition, the time delay cut off the short-lived background fluorescence [ 26 ].…”

Section: Resultsmentioning

confidence: 99%

“…In order to calculate the TagRFP fluorescence without the contribution of terbium fluorescence, it was necessary to carry out the titration of terbium-binding peptide linked to the TagRFP protein with photobleached chromophore using Tb(NO 3 ) 3 solution as described in [ 26 ]. The results of that titration are shown in Figure 6 .…”

Section: Resultsmentioning

confidence: 99%

“…As a result, we showed that there is an energy transfer in the Tb 3+ -TBP-19-TagRFP sensor from the terbium ion to the TagRFP chromophore. For the first time we obtained the spectrum of the sensitized TagRFP fluorescence with time delay for the previously described sensor [ 26 , 27 ]. Using the titration curve of TBP-19-TagRFP at 606 nm ( Figure 7 b), which was obtained by subtraction of intensity of the photobleached sample from the intensity of the corrected for reabsorption non-bleached sample, the dissociation constant of Tb 3+ and TBP-19-TagRFP complex was calculated.…”

Section: Resultsmentioning

confidence: 99%

“…Our laboratory has developed a genetically encoded FRET sensor for caspase-3 Tb 3+ -TBP-19-TagRFP [ 26 , 27 ], which consists of the complex of Tb 3+ with terbium-binding peptide YIDTNNDGWYEGDELLA [ 28 ] as a donor, flexible linker VDGGSGGDEVDGWGGSGLD [ 29 ] with the caspase-3 cleavage site DEVD [ 30 ], which was used earlier in the FRET-pair with a non-fluorescent acceptor [ 31 ], and red fluorescent protein TagRFP [ 26 , 32 ] as an acceptor. There are two donor-acceptor pairs in the resulting structure, which transfer energy using the fluorescence resonance mechanism.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

Goryashchenko

Khrenova

Bochkova

et al. 2015

IJMS

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

Goryashchenko

Khrenova

Bochkova

et al. 2015

IJMS

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“…These include a study of the intra-and intermolecular distances in macromolecules (Vazquez-Ibar et al, 2002), and a study on protein-peptide interactions (Sculimbrene and Imperiali, 2006). More recently, Arslanbaeva et al have used LBPs and fluorescent proteins as FRET pairs, and proposed their use in time-resolved fluorescence (TRF) applications (Arslanbaeva et al, 2010;Arslanbaeva et al, 2011). Using this approach, there is considerable potential for the LBPs in bioaffinity assays and medical diagnostics.…”

Section: Lanthanide-binding Peptidesmentioning

confidence: 99%

Luminescent lanthanide reporters: new concepts for use in bioanalytical applications

Vuojola

Soukka

2014

Methods Appl. Fluoresc.

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Lanthanides represent the chemical elements from lanthanum to lutetium. They intrinsically exhibit some very exciting photophysical properties, which can be further enhanced by incorporating the lanthanide ion into organic or inorganic sensitizing structures. A very popular approach is to conjugate the lanthanide ion to an organic chromophore structure forming lanthanide chelates. Another approach, which has quickly gained interest, is to incorporate the lanthanide ions into nanoparticle structures, thus attaining improved specific activity and a large surface area for biomolecule immobilization. Lanthanide-based reporters, when properly shielded from the quenching effects of water, usually express strong luminescence emission, multiple narrow emission lines covering a wide wavelength range, and exceptionally long excited state lifetimes enabling time-gated luminescence detection. Because of these properties, lanthanide-based reporters have found widespread applications in various fields of life. This review focuses on the field of bioanalytical applications. Luminescent lanthanide reporters and assay formats utilizing these reporters pave the way for increasingly sensitive, simple, and easily automated bioanalytical applications.

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Genetically Encoded Protease Substrate Based on Lanthanide-Binding Peptide for Time-Gated Fluorescence Detection

et al. 2013

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The study of biomolecular interactions is at the heart of biomedical research. Fluorescence and Förster resonance energy transfer (FRET) are potent and versatile tools in studying these interactions. Fluorescent proteins enable genetic encoding which facilitates their use in recombinant protein and in vivo applications. To eliminate the autofluorescence background encountered in applications based on fluorescent proteins, lanthanide labels can be used as donor fluorophores. Their long emission lifetime enables the use of time-gating that significantly improves assay sensitivity. In this work, we have combined the favorable characteristics of a terbium-ion-containing lanthanide-binding peptide (Tb(3+)-LBP) and green fluorescent protein (GFP) in a FRET-based homogeneous protease activity assay. The used genetically engineered construct had LBP and GFP sequences at adjacent ends of a linker that encoded the recognition sequence for caspase-3. Caspase proteases are central mediators in apoptosis and, consequently, are of great interest in the pharmaceutical industry. The designed fluorogenic protease substrate was applied for the detection of caspase-3 activity. We were able to demonstrate, for the first time, the applicability of a Tb(3+)-LBP-GFP energy-transfer pair in a protease activity assay. The intrinsically fluorescent and genetically encodable components enable easy expression of the construct without the need of cumbersome chemical labeling. By varying the fluorescent protein and the protease specificity of the internal linker sequence, the method can be applied for the detection of a wide variety of proteases.

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Induction-resonance energy transfer between the terbium-binding peptide and the red fluorescent proteins DsRed2 and TagRFP

Cited by 4 publications

References 24 publications

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

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

Luminescent lanthanide reporters: new concepts for use in bioanalytical applications

Genetically Encoded Protease Substrate Based on Lanthanide-Binding Peptide for Time-Gated Fluorescence Detection

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