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Functional complementation of high-efficiency resonance energy transfer: a new tool for the study of protein binding interactions in living cells.
| Content Provider | Semantic Scholar |
|---|---|
| Author | Molinari, Paola Casella, Ida |
| Copyright Year | 2008 |
| Abstract | Green bioluminescence in Renilla species is generated by a approximately 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 beta-arrestins. We show that complementation-induced BRET allows detection of the GPCR-beta-arrestin interaction in a simple luminometric assay with high signal-to-noise ratio, good dynamic range and rapid response. |
| File Format | PDF HTM / HTML |
| Alternate Webpage(s) | http://www.biochemj.org/content/ppbiochemj/409/1/251.full.pdf |
| PubMed reference number | 17868039v1 |
| Volume Number | 409 |
| Issue Number | 1 |
| Journal | The Biochemical journal |
| Language | English |
| Access Restriction | Open |
| Subject Keyword | Arrestins Beta-Arrestin-1, human Bioluminescence test Energy Transfer Green Fluorescent Proteins Luciferases Luciferases, Renilla Polypeptides Renilla (invertebrate) SAG gene beta-Arrestins coelenterazine |
| Content Type | Text |
| Resource Type | Article |
