Photochromic FRET Sensors to Monitor Heme Protein Dynamics

Abstract

The dynamic imaging of redox proteins is crucial for understanding their role in cell signaling. The spatiotemporal monitoring would provide an insight on regulation as well as determining endogenous factors directly affecting oxidation and reduction. However, limited number of probes and methods are available to measure dynamics with high specificity and sensitivity in living species. C-type cytochromes (Cyt c) are an important class of redox proteins whose function plays a key role for electron transfer in mitochondria and triggering apoptosis. Here we developed a new protein biosensor using Cyt c that is linked to a fluorescent reporter protein. The redox state of Cyt c was measured reversibly by photochromic fluorescence resonance energy transfer (pcFRET). The changes in the absorption of the endogenous heme cofactor bound to Cyt c covalently can modulate the fluorescence emission of the reporter. The multiple redox cycle of Cyt c was clearly monitored by pcFRET. We also demonstrated that the reporter could be used to monitor redox activity of Cyt c in living E. coli.