Chemistry & Biology
Volume 19, Issue 3, 23 March 2012, Pages 353-360
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Article
A Fluorogenic Red Fluorescent Protein Heterodimer

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Summary

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 Kd 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.

Highlights

► A dimerization-dependent red fluorescent protein (ddRFP) was engineered ► ddRFP fluorogenesis results from modulation of pKa and quantum yield ► ddRFP-based biosensors exhibit reversible intensiometric responses ► ddRFP-based biosensors can detect Ca2+ dynamics and caspase-3 activity in live cells

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