(LEFT) This Cdc42 biosensor was constructed from a fragment of WASP that binds selectively to activated Cdc42 (due to the green CRIB domain). The WASP fragment was derivatized with an environment-sensing dye that changed fluorescence when the biosensor bound Cdc42. Dye at position 271 (red) was in a hydrophobic pocket formed by amino acids from both WASP (blue) and Cdc42 (orange). (RIGHT) A library of FN3 monobodies is screened to find a library member with the appropriate binding selectivity and affinity for the targeted protein state. The library is based on a uniform scaffold stable in living cells and suitable for conversion to biosensors. The appropriate library member is derivatized with an environmentally sensitive dye to report target binding. Here, binding to active Src family kinases leads to increased fluorescence from a merocyanine dye.
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~ Updated 04/08/2