Mutational analysis of a red fluorescent protein-based calcium ion indicator

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dc.contributor.authorCampbell, Robert E.
dc.contributor.authorCarlson, Haley
dc.date.accessioned2014-10-08
dc.date.accessioned2022-05-27T01:13:16Z
dc.date.available2022-05-27T01:13:16Z
dc.date.issued2013
dc.description.abstractAs 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.
dc.format.extent1.83 mb
dc.format.mimetypePDF
dc.identifier.citationCarlson, 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
dc.identifier.doihttps://doi.org/10.3390/s130911507
dc.identifier.urihttps://hdl.handle.net/20.500.14078/166
dc.languageEnglish
dc.language.isoen
dc.rightsAttribution (CC BY)
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/
dc.subjectred fluorescent protein
dc.subjectcalcium ion
dc.subjectgenetically encoded sensor
dc.subjectmutagenesis
dc.subjectspectroscopy
dc.titleMutational analysis of a red fluorescent protein-based calcium ion indicator
dc.typeArticle
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