Simple Way to Detect Trp to Tb3+ Resonance Energy Transfer in Calcium-Binding Peptides Using Excitation Spectrum
Status PubMed-not-MEDLINE Language English Country Netherlands Media print-electronic
Document type Journal Article
Grant support
SVV 260426
Ministerstvo Školství, Mládeže a Tělovýchovy
354611
Grantová Agentura, Univerzita Karlova
PubMed
30471022
DOI
10.1007/s10895-018-2326-0
PII: 10.1007/s10895-018-2326-0
Knihovny.cz E-resources
- Keywords
- Calcium-binding site, Energy transfer, Excitation Spectrum, Protein fluorescence, Terbium phosphorescence, Tryptophan,
- Publication type
- Journal Article MeSH
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.
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