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Chem Biol. 2012 Mar 23;19(3):353-60. doi: 10.1016/j.chembiol.2012.01.006.

A fluorogenic red fluorescent protein heterodimer.

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1
Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada.

Abstract

The expanding repertoire of genetically encoded biosensors constructed from variants of Aequorea victoria green fluorescent protein (GFP) enable the imaging of a variety of intracellular biochemical processes. To facilitate the imaging of multiple biosensors in a single cell, we undertook the development of a dimerization-dependent red fluorescent protein (ddRFP) that provides an alternative strategy for biosensor construction. An extensive process of rational engineering and directed protein evolution led to the discovery of a ddRFP with a K(d) of 33 μM and a 10-fold increase in fluorescence upon heterodimer formation. We demonstrate that the dimerization-dependent fluorescence of ddRFP can be used for detection of a protein-protein interaction in vitro, imaging of the reversible Ca²⁺-dependent association of calmodulin and M13 in live cells, and imaging of caspase-3 activity during apoptosis.

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PMID:
22444590
PMCID:
PMC3560288
DOI:
10.1016/j.chembiol.2012.01.006
[Indexed for MEDLINE]
Free PMC Article

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