Simple Way to Detect Trp to Tb3+ Resonance Energy Transfer in Calcium-Binding Peptides Using Excitation Spectrum
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 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 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 Tb3+ 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.