As part of an ongoing effort to develop genetically encoded calcium ion (Ca2+
) indicators we recently described a new variant, designated CH-GECO2.1, that is a genetic chimera of the red fluorescent protein (FP) mCherry, calmodulin (CaM), and a peptide that binds to Ca2+
-bound CaM. In contrast to the closely related Ca2+
indicator R-GECO1, CH-GECO2.1 is characterized by a much higher affinity for Ca2+
and a sensing mechanism that does not involve direct modulation of the chromophore pKa
. To probe the structural basis underlying the differences between CH-GECO2.1 and R-GECO1, and to gain a better understanding of the mechanism of CH-GECO2.1, we have constructed, purified, and characterized a large number of variants with strategic amino acid substitutions. This effort led us to identify Gln163 as the key residue involved in the conformational change that transduces the Ca2+
binding event into a change in the chromophore environment. In addition, we demonstrate that many of the substitutions that differentiate CH-GECO2.1 and R-GECO1 have little influence on both the Kd
and the sensing mechanism, and that the interdomain linkers and interfaces play important roles.