Simple Way to Detect Trp to Tb 3+ Resonance Energy Transfer in Calcium-Binding Peptides Using Excitation Spectrum.

Autor: Lišková P; Department of Genetics and Microbiology, Faculty of Science, Charles University, Viničná 5, 128 43, Prague 2, Czech Republic., Konopásek I; Department of Genetics and Microbiology, Faculty of Science, Charles University, Viničná 5, 128 43, Prague 2, Czech Republic., Fišer R; Department of Genetics and Microbiology, Faculty of Science, Charles University, Viničná 5, 128 43, Prague 2, Czech Republic. radovan.fiser@natur.cuni.cz.
Jazyk: angličtina
Zdroj: Journal of fluorescence [J Fluoresc] 2019 Jan; Vol. 29 (1), pp. 9-14. Date of Electronic Publication: 2018 Nov 23.
DOI: 10.1007/s10895-018-2326-0
Abstrakt: The sensitized phosphorescence of Tb 3+ is often used for the assessment of the ion binding to various chelating agents or natural Ca 2+ -binding proteins. The detailed structure of the Tb 3+ excitation spectrum gives a special advantage for analysis; any extra absorption peak can be easily detected which provides simple and direct evidence that resonance energy transfer occurs. By employing the Tb 3+ phosphorescence, we characterized the Ca 2+ -binding sites of two related peptides - self-processing module of the FrpC protein produced by bacterium Neisseria meningitidis and the shorter peptide derived from FrpC. Here we show that while the increase of direct Tb 3+ excitation at 243 nm generally corresponds to Tb 3+ association with various binding sites, the excitation enhancement in the 250-300 nm band signifies Tb 3+ -binding in the close proximity of aromatic residues. We demonstrate that the presence of resonance energy transfer could be easily detected by inspecting Tb 3+ excitation spectra. Additionally, we show that the high level of specificity of Tb 3+ steady state detection on the spectral level could be reached at very low Tb 3+ concentrations by taking advantage of its narrow phosphorescence emission maximum at 545 nm and subtracting the averaged autofluorescence intensities outside this peak, namely at 525 and 565 nm.
Databáze: MEDLINE
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