Abstract
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.
Highlights
Molecular sensors that enable non-invasive fluorescence imaging of intracellular Ca2+ dynamics with high spatial and temporal resolution are powerful tools in modern cell biology and neuroscience research
CH-GECO2.1 and R-GECO1, and to obtain insight into the mechanism of CH-GECO2.1, we report the characterization of a barrage of single-site mutants of CH-GECO2.1
Further reversion of a mutation close to the fluorescent protein (FP)-CaM interface (Asp191Gly) and one final mutation in CaM that is relatively distant from the EF hands (Asn109Asp) produced a protein with a Kd of 35 nM (Figure 3E). These results clearly demonstrate that the dramatic difference in Kd between R-GECO1 and CH-GECO2.1 cannot be fully explained by the mutations within the CaM domain
Summary
Molecular sensors that enable non-invasive fluorescence imaging of intracellular Ca2+ dynamics with high spatial and temporal resolution are powerful tools in modern cell biology and neuroscience research. While organic dye-based Ca2+ indicators have long been a mainstay of such research [3], over the last decade proteinaceous Ca2+ indicators have emerged as a preferred alternative for many applications. The major advantage of proteinaceous Ca2+ indicators is that they are genetically encodable and can be tissue-selectively expressed and imaged in transgenic model organisms [4]. The development of proteinaceous Ca2+ indicators became a possibility only after the discovery, cloning, and subsequent optimization of the Aequorea victoria green FP [5]. Due to its inherent ability to generate a chromophore through an autonomous series of post-translational modifications, the green
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