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Article Dans Une Revue Biochemical Journal Année : 2007

Functional complementation of high efficiency resonance energy transfer. A new tool for the study of protein binding interactions in living cells

Paola Molinari
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Ida Casella
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Tommaso Costa
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Résumé

Green bioluminescence in Renilla species is generated by a ~ 100% efficient resonance energy transfer (RET) process that is caused by the direct association of a blue-emitting luciferase (Rluc) and a green fluorescent protein (RGFP). Despite the high efficiency, such system was never evaluated as a potential reporter of protein-protein interactions. To address the question, we compared and analyzed in mammalian cells the bioluminescence of Rluc and RGFP co expressed as free native proteins, or as fused single-chain polypeptides and tethered partners of self-assembling coiled-coils. Here we show that: (a) No spontaneous interactions generating detectable BRET signals occur between the free native proteins. (b) High efficiency BRET similar to that observed in Renilla occurs both in fusion proteins and self-interacting chimeras, but only if the amino terminal of RGFP is free. (c) The high efficiency BRET interaction is associated to a dramatic increase in light output when the luminescent reaction is triggered by low-quantum yield coelenterazine analogs. Here we propose a new functional complementation assay based on the detection of the high efficiency BRET signal that is generated when the reporters Rluc and RGFP are brought into close proximity by a pair of interacting proteins to which they are linked. To demonstrate its performance we implemented the assay to measure the interaction between G protein-coupled receptors (GPCRs) and β-arrestins. We show that complementation-induced BRET allows detecting the GPCR-β-arrestin interaction in a simple luminometric assay with high signal to noise ratio, good dynamic range, and rapid response.

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Dates et versions

hal-00478830 , version 1 (30-04-2010)

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Paola Molinari, Ida Casella, Tommaso Costa. Functional complementation of high efficiency resonance energy transfer. A new tool for the study of protein binding interactions in living cells. Biochemical Journal, 2007, 409 (1), pp.251-261. ⟨10.1042/BJ20070803⟩. ⟨hal-00478830⟩

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