Mutational analysis of a red fluorescent protein-based calcium ion indicator
Faculty Advisor
Date
2013
Keywords
red fluorescent protein, calcium ion, genetically encoded sensor, mutagenesis, spectroscopy
Abstract (summary)
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 for Ca2+ and the sensing mechanism, and that the interdomain linkers and interfaces play important roles.
Publication Information
Carlson, 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/s130911507
Notes
Item Type
Article
Language
English
Rights
Attribution (CC BY)
