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

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.