Redox status, which can vary over time and across subcellular compartments, is an important indicator of cell activity and health. Fluorescent protein sensors are particularly useful for the measurement of cellular redox dynamics because they are genetically encoded, they can be targeted to organelles, and they are typically non-cytotoxic. For example, the reduction-oxidation-sensitive green fluorescent proteins (roGFPs) are sensors that measure cellular redox and can detect reactive oxygen species (ROS). The roGFP sensors encode two cysteines engineered in close proximity to the chromophore, and a change in fluorescence is observed upon reduction of the disulfide bond formed by the cysteines. Currently, these sensors emit green fluorescence. In order to enable detection of varying ROS dynamics across cellular compartments, red emitting variants are needed. Here, we describe efforts to develop roGFPs with red-shifted emission via a Förster resonance energy transfer (FRET) relay strategy. Sensors were screened for FRET efficiency with steady-state and time-resolved fluorescence measurements. Functional sensors were characterized for efficient mitochondrial targeting and response to induced oxidative stress in live cells. We will present our progress towards demonstrating that these red-shifted sensors and the green fluorescent roGFPs can be used to simultaneously monitor redox dynamics in different cellular compartments.

Constructing red-shifted fluorescent protein sensors of cellular redox status

254th National Meeting (2017)

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