Green bioluminescence in Renilla species is generated by a ∼100% efficient RET (resonance energy transfer) process that is caused by the direct association of a blue-emitting luciferase [Rluc (Renilla luciferase)] and an RGFP (Renilla green fluorescent protein). Despite the high efficiency, such a system has never been evaluated as a potential reporter of protein–protein interactions. To address the question, we compared and analysed 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: (i) no spontaneous interactions generating detectable BRET (bioluminescence RET) signals occur between the free native proteins; (ii) high-efficiency BRET similar to that observed in Renilla occurs in both fusion proteins and self-interacting chimaeras, but only if the N-terminal of RGFP is free; (iii) the high-efficiency BRET interaction is associated with a dramatic increase in light output when the luminescent reaction is triggered by low-quantum yield coelenterazine analogues. 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 GPCRs (G-protein-coupled receptors) and β-arrestins. We show that complementation-induced BRET allows detection of the GPCR–β-arrestin interaction in a simple luminometric assay with high signal-to-noise ratio, good dynamic range and rapid response.
Functional complementation of high-efficiency resonance energy transfer: a new tool for the study of protein binding interactions in living cells
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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. Biochem J 1 January 2008; 409 (1): 251–261. doi: https://doi.org/10.1042/BJ20070803
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