Campbell, Robert E.Carlson, Haley2014-10-082022-05-272022-05-272013Carlson, H. J., & Campbell, R. E. (2013b). Mutational analysis of a red fluorescent protein-based calcium ion indicator. Sensors, 13(9), 11507–21. doi:10.3390/s130911507https://hdl.handle.net/20.500.14078/166As 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 for Ca2+ and the sensing mechanism, and that the interdomain linkers and interfaces play important roles.1.83 mbPDFenAttribution (CC BY)red fluorescent proteincalcium iongenetically encoded sensormutagenesisspectroscopyArticlehttps://doi.org/10.3390/s130911507