Figure 2:

Single FP fluorescent biosensor designs for cellular analytes and membrane potential.

Insertion of a sensing unit into a FP. Calmodulin, mEpac and mPDE5α undergo conformational changes in response to binding Ca^{2+ 144}, cAMP¹⁶³, and cGMP¹⁶⁴, respectively, which perturbs the chromophore and alters the fluorescence. Binding can either lead to an increase in fluorescence, as seen in the Ca²⁺ biosensor camgaroo1¹⁴⁴, or a decrease in fluorescence, as seen in the cAMP biosensor flamindo¹⁶³. B) Sandwiching a cpFP between sensing units. Biosensors have utilized sensing units that comprise either separate receiver and switch domains^148,150 or split proteins that re-constitute during protein folding^165–167. For example, GCaMP biosensors utilize separate domains of CaM and M13, where calcium binding to CaM promotes the binding of CaM to M13 and results in a conformational change that leads to an increase in GFP fluorescence. On the other hand, the membrane voltage sensor ASAP1 inserts cpGFP into the voltage-sensing domain of the chicken voltage-sensitive phosphatase Gg-VSP, which reconstitutes after folding and depolarization leads to a conformational change in the 4^th transmembrane segment that alters the fluorescence of cpGFP¹⁶⁷ C) Insertion of a FP into a voltage-sensitive channel. The conformational changes induced in voltage-sensitive K⁺ and Na⁺ channels alter the fluorescence of GFP to act as biosensors of membrane potential^168,169.