Imaging of conformational changes of proteins with a new environment/sensitive fluorescent probe designed for site specific labeling of recombinant proteins in live cells

We demonstrate herein a new method for imaging conformational changes of pr oteins in live cells using a new synthetic environment-sensitive fluorescen t probe, 9-amino-6,8-bis(1,3,2-dithioarsolan-2-yl)-5H-benzo[a]phenoxazin-5- one. This fluorescent probe can be attached to recombinant proteins contain ing four cysteine residues at the i, i + i, i + 4, and i + 5 positions of a n alpha -helix. The specific binding of the fluorescent probe to this 4Cys motif enables fluorescent labeling inside cells by its extracellular admini stration. The high sensitivity of the fluorophore to its environment enable s monitoring of the conformational changes of the proteins in live cells as changes in its fluorescence intensity, The present method was applied to c almodulin (CaM), a Ca2+-binding protein that was well known to expose hydro phobic domains, depending on the Ca2+ concentration. A recombinant CaM fuse d at its C-terminal with a helical peptide containing a 4Cys motif was labe led with the fluorescent probe inside live cells. The fluorescence intensit y changed reversibly depending on the intracellular Ca2+ concentration, whi ch reflected the conformational change of the recombinant CaM in the live c ells.