Dual-color click beetle luciferase heteroprotein fragment complementation assays.
Understanding the functional complexity of protein interactions requires mapping biomolecular complexes within the cellular environment over biologically relevant time scales. Herein, we describe a set of reversible multicolored heteroprotein complementation fragments based on various firefly and click beetle luciferases that utilize the same substrate, D-luciferin. Luciferase heteroprotein fragment complementation systems enabled dual-color quantification of two discrete pairs of interacting proteins simultaneously or two distinct proteins interacting with a third shared protein in live cells. Using real-time analysis of click beetle green and click beetle red luciferase heteroprotein fragment complementation applied to β-TrCP, an E3-ligase common to the regulation of both β-catenin and IκBα, GSK3β was identified as a candidate kinase regulating IκBα processing. These dual-color protein interaction switches may enable directed dynamic analysis of a variety of protein interactions in living cells.
Villalobos
Victor
V
Molecular Imaging Center, Mallinckrodt Institute of Radiology, Department of Developmental Biology, Washington University, St. Louis, MO 63110, USA.
Naik
Snehal
S
Bruinsma
Monique
M
Dothager
Robin S
RS
Pan
Mei-Hsiu
MH
Samrakandi
Mustapha
M
Moss
Britney
B
Elhammali
Adnan
A
Piwnica-Worms
David
D
D000818:Animals; D052160:Benzothiazoles / Q000737:chemistry; D002460:Cell Line; D001517:Coleoptera / Q000201:enzymology*; D003116:Color; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D008156:Luciferases / Q000737:chemistry* / Q000378:metabolism; D025941:Protein Interaction Mapping; D020123:Sirolimus / Q000494:pharmacology; D051176:beta Catenin / Q000378:metabolism; D044844:beta-Transducin Repeat-Containing Proteins / Q000378:metabolism
D052160:Benzothiazoles; C532924:D-luciferin; D007166:Immunosuppressive Agents; D051176:beta Catenin; D044844:beta-Transducin Repeat-Containing Proteins; D008156:Luciferases; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3; D020123:Sirolimus