title: Mutational analysis of a red fluorescent protein-based calcium ion indicator.
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.
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, AB, Canada. hcarlson@ualberta.ca
authors.forename: Haley J
authors.initials: HJ
authors.lastname: Carlson
authors.forename: Robert E
authors.initials: RE
authors.lastname: Campbell
mesh_terms: D002118:Calcium / Q000737:chemistry*; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D017930:Genes, Reporter / Q000235:genetics; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000379:methods*; D000097573:Red Fluorescent Protein
chemical_list: D002118:Calcium; D002147:Calmodulin; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein