Dual-color click beetle luciferase heteroprotein fragment complementation assays

Chem Biol. 2010 Sep 24;17(9):1018-29. doi: 10.1016/j.chembiol.2010.06.018.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Benzothiazoles / chemistry
  • Cell Line
  • Coleoptera / enzymology*
  • Color
  • Glycogen Synthase Kinase 3 / metabolism
  • Glycogen Synthase Kinase 3 beta
  • Humans
  • Immunosuppressive Agents / pharmacology
  • Luciferases / chemistry*
  • Luciferases / metabolism
  • Protein Interaction Mapping
  • Sirolimus / pharmacology
  • beta Catenin / metabolism
  • beta-Transducin Repeat-Containing Proteins / metabolism

Substances

  • Benzothiazoles
  • D-luciferin
  • Immunosuppressive Agents
  • beta Catenin
  • beta-Transducin Repeat-Containing Proteins
  • Luciferases
  • GSK3B protein, human
  • Glycogen Synthase Kinase 3 beta
  • Glycogen Synthase Kinase 3
  • Sirolimus