The dityrosine cross-link as an intrinsic donor for assembling FRET pairs in the study of protein structure

Dityrosine-Lucifer yellow (DT-LY) was used as the donor-acceptor pair in studies of the topography of juvenile hormone‐binding protein (JHBP). The Förster distance, R0 = 30.5 Å for DT-LY was determined. Separation distances (R) between DT and the fluorescent probes placed at the 219 and 224 position were 26 and 28 Å and correspond to that found from X-ray analysis (23 and 24 Å, respectively). Higher than expected efficiency of energy transfer between DT and LY probe placed in position 171 was observed, indicating that this probe is immobilized in the protein structure (κ2 = 3.25). Red-edge excitation shift (REES) analysis supported this assumption. Slight changes in Förster resonance energy transfer (FRET) efficiency were observed after incubating the labeled proteins with juvenile hormone III (JH III). This is the first report showing the application of DT fluorescence for the analysis of protein conformation.

Graphical abstractDownload full-size imageHighlights► Dityrosine and Lucifer yellow (LY) were applied for analysis of JHBP structure. ► The separation distances between the fluorophores were compared with X-ray data. ► LY moiety in position 171 is embedded in the protein molecule structure.