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A fluorogenic red fluorescent protein heterodimer.

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 ?M 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.

SUBMITTER: Alford SC

PROVIDER: S-EPMC3560288 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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A fluorogenic red fluorescent protein heterodimer.

Alford Spencer C SC Abdelfattah Ahmed S AS Ding Yidan Y Campbell Robert E RE

Chemistry & biology 20120301 3

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 ev ...[more]

PMID: 22444590

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