title: 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 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.
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada.
authors.forename: Spencer C
authors.initials: SC
authors.lastname: Alford
authors.forename: Ahmed S
authors.initials: AS
authors.lastname: Abdelfattah
authors.forename: Yidan
authors.initials: Y
authors.lastname: Ding
authors.forename: Robert E
authors.initials: RE
authors.lastname: Campbell
mesh_terms: D017209:Apoptosis; D015374:Biosensing Techniques; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D002118:Calcium; D002147:Calmodulin; D053148:Caspase 3; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D000097573:Red Fluorescent Protein