Bacteria Luciferase-based Bret Technology For Detection Of Protein-protein Interactions

Protein-protein interactions are important for virtually every biological process and a number ofelegant approaches have been designed to detect and evaluate such interactions. However, few of thesemethods allow the detection of dynamic and real-time protein-protein interactions in bacteria.Here we describe a bioluminescence resonance energy transfer (BRET) system based on the bacterialluciferase LuxAB. We found that the spectrum of LuxAB emission is sufficiently broad to excitefluorescence of eYFP but not that of enhanced GFP (eGFP) or mOrange, a fluorescent protein fromDiscosoma sp. The eYFP accepts the emission from LuxAB and emits yellow fluorescence. Importantly,BRET occurs when LuxAB and eYFP fused respectively to the interacting protein pair FlgM and FliA.Further we observed rapamycin inducible interactions between FRB and FKBP12and a dose-dependentabolishment of such interactions by FK506, the ligand of FKBP12. Importantly, the use of FK506to reversesirolimus-mediated binding between FRB and FKBP allowed us to obtain an IC50value for the competitiveinhibitor. Using this system, we showed that osmotic stress or low pH efficiently induced multimerization ofthe regulatory protein OmpR, and that the multimerization induced by low pH can be reversed by aneutralizing agent, further indicating the usefulness of this system in the measurement of dynamicinteractions. This method can be adapted to analyze dynamic protein-protein interactions and the importanceof such interactions in bacterial processes as development and pathogenicity. The BRET system based onbacterial luciferase LuxAB is the first time, as we known, to measure real-time dynamic protein-proteininteractions in prokaryotic cells.