Circular Permutation Probes for Illuminating Phosphorylation of Estrogen Receptor
The present protocol demonstrates a new strategy for imaging ligand-triggered protein phosphorylation using circularly permutated luciferases (cpLuc): (1) a luciferase is first fragmented into two segments for creating new N- and C-terminal ends in the hydrophilic region, (2) the original N- and C-terminal ends are circularly permutated and linked via a GS linker, whereas the new ends made by fragmentation are correspondingly linked with two proteins of interest. When the new ends of the cpLuc are linked with the ligand-binding domain of estrogen receptor (ER LBD) and Src homology two domain of Src (SH2), the estrogen can trigger phosphorylation of the ER LBD and consequent intramolecular ER LBD–SH2 binding. This interaction triggers an approximation of the adjacent fragments of split-cpLuc recovering the enzyme activity. This probe design greatly improves signal-to-noise (S/N) ratios upon tracing weak protein–protein interactions (PPIs) in mammalian cells.
Key wordsCircular permutation Phosphorylation Luciferase Estrogen receptor Bioluminescence
This work was supported by grants from Japan Society for the Promotion of Science (JSPS), grant numbers 26288088, 16K14051, and 15KK0029.