Engineering A Hybrid FRET Biosensor to Study Proteolytic Activities of MT1‐MMP

The genetically‐encoded biosensors based on Förster resonance energy transfer (FRET) are extensively used for real‐time monitoring of biological processes including enzymatic activities in living cells with high spatiotemporal resolutions. However, the development of a membrane‐targeted FRET biosensor remains a challenge partly due to the poor efficiency of transportation and integration of fusion proteins at the plasma membrane, which results in high background signals. To overcome this deficiency, we introduced an alternative strategy that allows the direct assembly of FRET biosensors in situ at the cell surface by integrating an enhanced cyan fluorescence protein (ECFP; a donor) and a fluorescent dye, R‐Phycoerythrin (R‐PE; an acceptor), for FRET action. We utilized the directed evolution technology and sequence‐function analysis to engineer a monobody into an efficient R‐PE binder, PEbody, to directly capture R‐PE. Our hybrid FRET biosensor was used to monitor the enzymatic activity of the membrane type 1 metalloproteinase (MT1‐MMP) on the extracellular surface of live cancer cells. The results revealed a cytoskeleton‐dependent distribution of MT1‐MMP activities at different cell‐cell contacts, with high and low activities at loose and stable cell‐cell contacts, respectively. Therefore, integrating the directed evolution approach and rational design can be employed to systematically design novel FRET biosensors capable of monitoring the molecular dynamics at the surface and other subcellular regions in living cells. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .