A Fluorogenic Red Fluorescent Protein Heterodimer

SummaryThe 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.

Graphical AbstractFigure optionsDownload high-quality image (517 K)Download as PowerPoint slideHighlights
► 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