DETECTION IN LIVING CELLS OF CA2-DEPENDENT CHANGES IN THE FLUORESCENCE EMISSION OF AN INDICATOR COMPOSED OF 2 GREEN FLUORESCENT PROTEIN VARIANTS LINKED BY A CALMODULIN-BINDING SEQUENCE - A NEW CLASS OF FLUORESCENT INDICATORS()

We have designed a novel fluorescent indicator composed of two green f luorescent protein variants joined by the calmodulin-binding domain fr om smooth muscle myosin light chain kinase. When (Ca2+)(4)-calmodulin is bound to the indicator (K-d = 0.4 nM), fluorescence resonance energ y transfer between the two fluorophores is attenuated; the ratio of th e fluorescence intensity measured at 505 nm to the intensity measured at 440 nm decreases B-fold. Images of microinjected living cells demon strate that emission ratios can be used to monitor spatio temporal cha nges in the fluorescence of the indicator, Changes in indicator fluore scence in these cells are coupled with no discernible lag(<1 s) to cha nges in the cytosolic free Ca2+ ion concentration, ranging from below 50 nM to similar to 1 mu M. This observation suggests that the activit y of a calmodulin target with a typical 1 nM affinity for (Ca2+)(4)-ca lmodulin is responsive to changes in the intracellular Ca2+ concentrat ion over the physiological range. It is likely that the indicator we d escribe can be modified to detect the levels of ligands and proteins i n the cell other than calmodulin.