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Sensors 2013, 13(9), 11507-11521; doi:10.3390/s130911507
Article

Mutational Analysis of a Red Fluorescent Protein-Based Calcium Ion Indicator

 and *
Received: 11 August 2013; in revised form: 27 August 2013 / Accepted: 29 August 2013 / Published: 2 September 2013
(This article belongs to the Special Issue Fluorescent Biosensors)
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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.
Keywords: red fluorescent protein; calcium ion; genetically encoded sensor; mutagenesis; spectroscopy red fluorescent protein; calcium ion; genetically encoded sensor; mutagenesis; spectroscopy
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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MDPI and ACS Style

Carlson, H.J.; Campbell, R.E. Mutational Analysis of a Red Fluorescent Protein-Based Calcium Ion Indicator. Sensors 2013, 13, 11507-11521.

AMA Style

Carlson HJ, Campbell RE. Mutational Analysis of a Red Fluorescent Protein-Based Calcium Ion Indicator. Sensors. 2013; 13(9):11507-11521.

Chicago/Turabian Style

Carlson, Haley J.; Campbell, Robert E. 2013. "Mutational Analysis of a Red Fluorescent Protein-Based Calcium Ion Indicator." Sensors 13, no. 9: 11507-11521.



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