Many fluorescent protein (FP)-based biosensors enable imaging of biochemical processes in live cells. However, red FP-based biosensors are limited. To address this deficiency, a dimerization-dependent red FP (ddRFP) was developed to provide an alternative template for biosensor construction. Extensive directed protein evolution led to the discovery of a ddRFP that exhibits a 10-fold increase in fluorescence upon heterodimerization. The reversible changes in fluorescent intensity that result from association of the ddRFP partners, or the irreversible decrease that accompanies dissociation of covalently linked partners following linker cleavage, provides a useful spectroscopic signal for biosensing applications. The utility of ddRFP is illustrated with a series of representative examples that include imaging of the Ca2+-dependent association of calmodulin and M13 in live cells, as well as the imaging of caspase-3 activity during apoptosis. This series of applications illustrates that ddRFP provides a general solution to the challenge of constructing intensiometric red fluorescent biosensors.

Fluorogenic red fluorescent protein heterodimers: A new template for live cell biosensor design

243rd National Meeting (2012)

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