Simple Way to Detect Trp to Tb3+ Resonance Energy Transfer in Calcium-Binding Peptides Using Excitation Spectrum
Autor: | Ivo Konopásek, Petra Liskova, Radovan Fišer |
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Rok vydání: | 2018 |
Předmět: |
chemistry.chemical_classification
Sociology and Political Science 010405 organic chemistry Chemistry Clinical Biochemistry Tryptophan Analytical chemistry Resonance Peptide 010402 general chemistry 01 natural sciences Biochemistry 0104 chemical sciences Clinical Psychology Ion binding Binding site Absorption (electromagnetic radiation) Phosphorescence Law Spectroscopy Social Sciences (miscellaneous) Excitation |
Zdroj: | Journal of Fluorescence. 29:9-14 |
ISSN: | 1573-4994 1053-0509 |
Popis: | The sensitized phosphorescence of Tb3+ is often used for the assessment of the ion binding to various chelating agents or natural Ca2+-binding proteins. The detailed structure of the Tb3+ 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 Tb3+ phosphorescence, we characterized the Ca2+-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 Tb3+ excitation at 243 nm generally corresponds to Tb3+ association with various binding sites, the excitation enhancement in the 250–300 nm band signifies Tb3+-binding in the close proximity of aromatic residues. We demonstrate that the presence of resonance energy transfer could be easily detected by inspecting Tb3+ excitation spectra. Additionally, we show that the high level of specificity of Tb3+ steady state detection on the spectral level could be reached at very low Tb3+ 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: | OpenAIRE |
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