Dual-Color Click Beetle Luciferase Heteroprotein Fragment Complementation Assays

Abstract

Summary Understanding the functional complexity of protein interactions requires mapping biomolecular complexes within the cellular environment over biologically-relevant time scales. Herein we describe a novel 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 discreet 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 novel 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.

Adaptive Mutations in the Nuclear Export Protein of Human-Derived H5N1 Strains Facilitate a Polymerase Activity-Enhancing Conformation

“…The luciferase activity observed at 37°C was set to 100%. Cell extracts containing N Luc -C Luc (N Luc and C Luc fused by linker sequence) served as a control for a temperature-stable luciferase (28). Error bars indicate standard deviations of three independent experiments.…”

“…To determine transient interactions between the N-and C-terminal moieties, HEK293T cells were transfected with plasmids encoding avian H5N1 NEP (avNEP) or huNEP fused to both N-and C-terminal click beetle luciferase moieties (N Luc -avNEP-C Luc , N Luc -huNEP-C Luc ), and cell extracts were prepared 24 h posttransfection at 4°C. To monitor temperature-dependent differences, these extracts were warmed up to 37°C for 30 min and subsequently cooled down to 34,31,28,25,22, and 19°C (20 min each temperature). At each temperature a fraction of the cell extract was used to determine the luciferase activity.…”

Interrogation of Inhibitor of Nuclear Factor κB α/Nuclear Factor κB (IκBα/NF-κB) Negative Feedback Loop Dynamics

“…later time point) TNF␣-induced ⌱B␣ degradation in governing ⌱B␣-FLuc resynthesis phase dynamics, we utilized a mutant bioluminescent reporter, B 5 3⌱B␣(S32A,S36A)-FLuc (35). The serine-to-alanine substitutions render ⌱B␣ unresponsive to IKK-directed phosphorylation and subsequent proteasomal degradation; however, the reporter is still responsive to the NF-B transcriptional activity elicited once endogenous ⌱B␣ is degraded and NF-B translocates into the nucleus.…”

Biosensors for Monitoring Autophagy

“…Cells would be otherwise non-fluorescent providing for a high signal-to-noise ratio. A similar and considerably more sensitive biosensor might be developed along similar lines if the FP is substituted for a member of the light-emitting luciferase family (Villalobos et al, 2010). Finally, it may be possible for an inactive pro-enzyme such as acid protease to be used to label targets.…”