Figure 3. Design of biosensors to screen for CaM binding sequences in GPER.

Biosensor is composed of a FRET donor ECFP and a FRET acceptor citrine EYFPc flanking each of the four submembrane domains in GPER and their truncated segments corresponding to our predicted CaM-binding sequences. The proximity between the donor and acceptor in the unbound state facilitate robust FRET when the donor ECFP is excited at 430 nm giving emission light at 475 nm, which in turn excites the acceptor EYFPc, which emits at 535 nm (A). Upon addition of CaM, if there is specific interaction with the GPER insert fragment, the conformational changes that occur upon specific interaction with CaM will disrupt FRET, decreasing emission from EYFPc while increasing that from donor ECFP. This signature spectral change identifies CaM binding (B). C and D, Typical spectrofluorometric response of biosensor in the absence of specific CaM binding to its linker (C), which corresponds to the biosensor configuration in (A), and in the presence of CaM and saturating concentration of Ca²⁺ (D), which corresponds to the biosensor configuration in (B).