Improving FRET Relay Mechanism for Redox Dynamics in Live Cells

Reactive oxygen species (ROS) have an important role with regards to intracellular signaling throughout the body. Levels of ROS fluctuate dynamically with differing sources of ROS as well as antioxidant mechanisms to regulate oxidative stress, cell to cell signaling, and cellular metabolism. In many cases, localized redox dynamics have not been elucidated due to a lack of tools available to study oxidative fluctuations localized to different compartments of ROS generation within cells. Previously, our lab reported the development of a new family of genetically encoded fluorescent protein redox biosensors. They utilize the ratiometric properties of roGFP2 and extend its fluorescence emission via Förester Resonance Energy Transfer (FRET) relay to an acceptor red fluorescent protein to enable dual-color imaging for live-cell microscopy. Here, a second generation of these proteins has been developed that has an increased FRET relay efficiency, obtained through decreasing the size of the linker length between the donor and acceptor fluorescent proteins. We characterized the redox and optical properties of the purified sensors, and expressed them into bacterial and mammalian cells to better understand the role of ROS within cellular signaling and metabolism