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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
*
Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada
* Author to whom correspondence should be addressed.
Received: 11 August 2013 / Revised: 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 (CC BY 3.0).

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

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