A bioluminescence resonance energy transfer (BRET) system: Application to interacting circadian clock proteins

Abstract

We describe a method for assaying protein interactions that offers some attractive advantages over previous assays. This method, called bioluminescence resonance energy transfer (BRET), uses a bioluminescent luciferase that is genetically fused to one candidate protein, and a green f luorescent protein mutant fused to another protein of interest. Interactions between the two fusion proteins can bring the luciferase and green f luorescent protein close enough for resonance energy transfer to occur, thus changing the color of the bioluminescent emission. By using proteins encoded by circadian (daily) clock genes from cyanobacteria, we use the BRET technique to demonstrate that the clock protein KaiB interacts to form homodimers. BRET should be particularly useful for testing protein interactions within native cells, especially with integral membrane proteins or proteins targeted to specific organelles.Interactions between proteins play a role in many biological processes. Current techniques to identify and characterize these interactions include in vitro-binding assays, library-based methods, and genetic methods (1). In this report, we introduce a method for assaying protein-protein interactions that takes advantage of a phenomenon that occurs in nature, namely, the Förster resonance energy transfer between a light-emitting luciferase and an acceptor fluorophore (2-5). The technique is related to an existing method for assessing protein-protein interaction, fluorescence resonance energy transfer (FRET). In this process, one fluorophore (the ''donor'') transfers its excited-state energy to another fluorophore (the ''acceptor''), which usually emits fluorescence of a different color. FRET efficiency depends on the spectral overlap, the relative orientation, and the distance between the donor and acceptor fluorophores. Generally, FRET occurs when the donor and acceptor are 10-100 Å apart (4), so it can be used to assay protein-protein proximity by attaching the donor and acceptor fluorophores to the candidate proteins. By using mutants of the green fluorescent protein (GFP; M r ϭ 27 kDa), it is possible to genetically attach donor and acceptor fluorophores to proteins (6-8). This GFP-based FRET assay allows protein interactions to be observed in the native organism under physiological conditions (9, 10). Moreover, compartmentalization of these interacting proteins is visible in the microscope (9, 10).As with any fluorescence technique, however, photobleaching and autofluorescence can limit the usefulness of FRET. It also can be complicated by direct excitation of the acceptor fluorophore. Furthermore, FRET may be impractical in tissues that are easily damaged by the excitation light or that are photoresponsive (e.g., retina). Our protein interaction assay, which we call bioluminescence resonance energy transfer (BRET), offers the advantages of FRET but avoids the consequences of fluorescence excitation. BRET is a naturally occurring phenomenon. For instance, when the photoprotein aequorin is purified from the...

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No Promoter Left Behind: Global Circadian Gene Expression in Cyanobacteria

“…This profound control of transcriptional activity in the cyanobacterium Synechococcus elongatus PCC 7942 is regulated by a molecular clockwork that is encoded by 3 genes-kaiA, kaiB, and kaiC-that form a cluster on the chromosome; inactivation of any of the kai genes abolishes clock function (Ishiura et al, 1998). The proteins encoded by these genes interact with one another (Iwasaki et al, 1999;Taniguchi et al, 2001;Xu et al, 1999) to form large complexes in vivo with KaiC as the core (Kageyama et al, 2003). KaiC exists in both phosphorylated and nonphosphorylated forms in vivo, and its phosphorylation status is correlated with clock speed (Nishiwaki et al, 2000;Iwasaki et al, 2002;Xu et al, 2003;Johnson, 2004).…”

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“…TAP1 is a transmembrane endoplasmic reticulum (ER) resident and was used as negative control (44). BRET is a very sensitive assay that allows the detection in living cells of interactions between two protein partners, one tagged with Rluc and one tagged with YFP (34,45,46). The nonradiative luminescence emitted by Rluc upon the addition of its substrate, coelenterazine, can excite the YFP if the two molecules are at a distance of 100 Å or less.…”