Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Record number 422745
Title Disentangling picosecond events that complicate the quantative use of the calcium sensor YC3.60
Author(s) Laptenok, S.; Stokkum, I.H.M. van; Borst, J.W.; Oort, B.F. van; Visser, A.J.W.G.; Amerongen, H. van
Source The Journal of Physical Chemistry Part B: Condensed Matter, Materials, Surfaces, Interfaces & Biophysical 116 (2012)9. - ISSN 1520-6106 - p. 3013 - 3020.
DOI https://doi.org/10.1021/jp211830e
Department(s) Biochemistry
Biophysics
Publication type Refereed Article in a scientific journal
Publication year 2012
Keyword(s) cyan fluorescent protein - resonance energy-transfer - time-resolved spectra - living cells - fret - photoconversion - ca2+ - yfp - spectroscopy - indicators
Abstract Yellow Cameleon 3.60 (YC3.60) is a calcium sensor based on Förster resonance energy transfer (FRET). This sensor is composed of a calmodulin domain and a M13 peptide, which are located in between enhanced cyan-fluorescent protein (ECFP) and the Venus variant of enhanced yellow-fluorescent protein (EYFP). Depending on the calcium concentration, the efficiency of FRET from donor ECFP to acceptor EYFP is changing. In this study, we have recorded time-resolved fluorescence spectra of ECFP, EYFP, and YC3.60 in aqueous solution with picosecond time resolution, using different excitation wavelengths. Detailed insight in the FRET kinetics was obtained by using global and target analyses of time- and wavelength-resolved fluorescence of purified YC3.60 in calcium-free and calcium-bound conformations. The results clearly demonstrate that for both conformations, there are two distinct donor populations: a major one giving rise to FRET and a minor one not able to perform FRET. The transfer time for the calcium-bound conformation is 21 ps, whereas it is in the order of 1 ns for the calcium-free conformation. Ratio imaging of acceptor and donor fluorescence intensities of YC3.60 is usually applied to measure Ca(2+) concentrations in living cells. From the obtained results, it is clear that the intensity ratio is strongly influenced by the presence of donor molecules that do not take part in FRET, thereby significantly affecting the quantitative interpretation of the results.
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