Sensors Based on Single cAMPBinding Domains

Crystallographic studies of cyclic nucleotide-binding domains from Epac and PKA revealed that cAMP binding induces conformational changes even in the absence of other parts of the protein (Diller et al. 2001; Rehmann et al. 2003). Based on these observations, a family of FRET sensor based on a single cAMP-binding domain from Epac 1, Epac 2, the PKA R subunit (Nikolaev et al. 2004) or a HCN channel

Fig. 3 Sensor based on a single cAMP-binding domain. (a) Schematic representation of the mechanism of action of Epac-camps and (b) schematic structure of Epac 1-camps

(Nikolaev et al. 2006) was generated. Such sensors have the advantage of an extremely simplified design with a single cAMP-binding domain sandwiched between YFP and CFP (Fig. 3), reducing in this way the chance to generate artifacts resulting from catalytic activity or potential interaction of the sensor with endogenous proteins. In addition, the sensitivity of such single-domain sensors proved to be reasonably high (0.9-5.9 |M) (Nikolaev et al. 2004), allowing the measurement of cAMP within the physiological range of concentrations. In addition, they show rapid cAMP-dependent FRET changes both in vitro and in intact cells, therefore allowing the achievement of high temporal resolution (Nikolaev et al. 2004).

0 0

Post a comment