Figure 1
Design of FRET and LRET Rac1 biosensors. (a) Schematic representation and (b) mammalian expression constructs of single-chain FRET or LRET Rac1 biosensors. A series of nine sensors was constructed in which alpha-helical ER/K linkers of different length (10 nm, 20 nm and 30 nm) were combined with three fluorophore pairs: (i) mCerulean and Ypet; (ii) circularly permutated mutant (cp227) of mTFP1 and circularly permutated mutant (cp229) of Venus; (iii) Tb(III) complex (bound to E. coli dihydrofolate reductase, eDHFR) and EGFP. (c) Sequence of a 30 nm alpha-helical ER/K linker (207 residues) that includes alternated repeats of approximately four negatively charged glutamates (red) and four negatively charged arginines or lysines (blue). The linker is rigid to keep protein components far apart in OFF state. Stochastic breaks allow protein interaction in the biosensor ON-state and promote a large change in donor-sensitized acceptor emission.
