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

The sensitized phosphorescence of Tb is often used for the assessment of the ion binding to various chelating agents or natural Ca-binding proteins. The detailed structure of the Tb 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 phosphorescence, we characterized the Ca-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 excitation at 243 nm generally corresponds to Tb association with various binding sites, the excitation enhancement in the 250-300 nm band signifies Tb-binding in the close proximity of aromatic residues. We demonstrate that the presence of resonance energy transfer could be easily detected by inspecting Tb excitation spectra. Additionally, we show that the high level of specificity of Tb steady state detection on the spectral level could be reached at very low Tb 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.