Biomedical research institute
     

    BRET (Bioluminescence Resonance Energy Transfer)

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    Protein interaction networks are of major importance in higher organisms. One of the perhaps most surprising findings of the genome-sequencing projects was the relatively low number of genes and their similar number in very different organisms [1]. This led to the suggestion that the biological complexity of organisms is not reflected by the number of genes but rather by the number of physiologically relevant protein interactions. Several techniques are able to detect protein-protein interactions in a dynamic manner. Among these is the BRET (Bioluminescence Resonance Energy Transfer) technique, which monitors protein interactions and its modulation (i.e. conformational changes) in living cells.

     

    Image1pourBRET 

     

    BRET occurs naturally in some marine species (for instance in the sea pansy Renilla reniformis) where a non-radiative energy transfer is observed between the Renilla luciferase (energy donor) and the Renilla green fluorescent protein (energy acceptor) (Fig. 1) [2]. In 1999, Xu et al. applied BRET to the detection of protein-protein interactions [3]. To reach this goal, one protein is fused to the donor and the other to the acceptor. Addition of coelenterazine, the natural substrate of Renilla luciferase (Rluc), leads to the emission of blue light (480 nm). To fulfill the conditions for energy transfer, the emission spectrum of the donor must overlap with the excitation spectrum of the acceptor. The typical energy acceptor used in BRET experiments, which fulfills these criteria, is the yellow fluorescent protein (YFP). If the two fusion proteins do not interact, only blue light is emitted upon substrate addition (Fig. 2). If the two fusion proteins interact and position the energy donor and acceptor within a distance less than 10 nm, a resonance energy transfer occurs and an additional light signal corresponding to the acceptor reemission can be detected (535 nm in the case of YFP, BRET1 version). In addition, BRET depends on the relative orientation of BRET partners due to the dipole-dipole nature of the resonance energy transfer mechanism. However, this parameter is often difficult to evaluate and becomes less important if the energy donor and acceptor are very mobile.
    In the BRET2 assay, the YFP is replaced by GPF2, a GFP mutant that can be excited at 400 nm, and the Rluc substrate is the Deepblue C (also known as coelenterazine 400a). The advantage of BRET2 is a superior separation of donor and acceptor peaks. Indeed, in the presence of Deepblue C, Rluc emits light at 400 nm, a wavelength that excites GFP2, which, in turn, emits light at 510 nm. The disadvantage of BRET2, compared to BRET1 is the 100-300 times lower intensity of emitted light as illustrated in Figure 2.                            
        

                     BRET

     

     Description of BRET1 and BRET2assays.



    BRET can be measured with a microscopic setting but is most often measured with a microplate reader. Since the description of the initial BRET version several other versions have been developed for review see [4, 5]. We recently proposed an improved BRET version, which is more sensitive and based on new donor/acceptor combinations such as Rluc8/YPet and Rluc8/RGFP [6].

    Our group developed several different BRET assays such as the BRET donor saturation and BRET competition assay to discriminate between ligand-induced recruitment and ligand-induced conformational changes and to assess the specificity of BRET signals [7-10].

    We regularly contribute to the promotion of the BRET technique by writing technical and review articles [5, 11-17] and by participating/organizing different Inserm Workshops and Training.


    References :

    [1] Lander, E. S., Linton, L. M., Birren, B., Nusbaum, C., et al., Initial sequencing and analysis of the human genome. Nature 2001, 409, 860-921.

    [2] Ward, W. W., Cormier, M. J., An energy transfer protein in coelenterate bioluminescence. Characterization of the Renilla green-fluorescent protein. J Biol Chem 1979, 254, 781-788.

    [3] Xu, Y., Piston, D. W., Johnson, C. H., A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins. Proc Natl Acad Sci U S A 1999, 96, 151-156.

    [4] Pfleger, K. D., Eidne, K. A., Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET). Nat Methods 2006, 3, 165-174.

    [5] Bacart, J., Corbel, C., Jockers, R., Bach, S., Couturier, C., The BRET technology and its application to screening assays. Biotechnol J 2008, 3, 311-324.

    [6] Kamal, M., Marquez, M., Vauthier, V., Leloire, A., et al., Improved donor/acceptor BRET couples for monitoring ß-arrestin recruitment to G protein-coupled receptors. Biotechnol J 2009, 4:1337-1344.


    [7] Ayoub, M. A., Couturier, C., Lucas-Meunier, E., Angers, S., et al., Monitoring of ligand-independent dimerization and ligand-induced conformational changes of melatonin receptors in living cells by bioluminescence resonance energy transfer. J Biol Chem 2002, 277, 21522-21528.

    [8] Couturier, C., Jockers, R., Activation of leptin receptor by a ligand-induced conformational change of constitutive receptor dimers. J. Biol. Chem. 2003, 278, 26604-26611.

    [9] Ayoub, M. A., Levoye, A., Delagrange, P., Jockers, R., Preferential formation of MT1/MT2 melatonin receptor heterodimers with distinct ligand interaction properties compared with MT2 homodimers. Mol Pharmacol 2004, 66, 312-321.

    [10] Levoye, A., Dam, J., Ayoub, M. A., Guillaume, J. L., et al., The orphan GPR50 receptor specifically inhibits MT(1) melatonin receptor function through heterodimerization. EMBO J 2006, 25, 3012-3023.

    [11] Boute, N., Jockers, R., Issad, T., The use of resonance energy transfer in high-throughput screening : BRET versus FRET. Trends Pharmacol Sci 2002, 23, 351-354.

    [12] Issad, T., Jockers, R., Bioluminescence Resonance Energy Transfer (BRET) to monitor protein-protein interactions, Human Press Inc, Totowa, NJ 2005.

    [13] Jockers R., Bouvier M and Marullo S. : Energy transfer-based approaches to study G protein-coupled receptor dimerization and activation. In The Encyclopedia of Genetics, Genomics,  Proteomics and Bioinformatics, ISBN : 0-470-84974-6, J.B. Jorde, P.F.R. Little, M.J. Dunn and S. Subramaniam, Eds., John Wiley & Sons, Ltd. (2005).

    [14] Jockers R., and Marullo S. : Basic techniques : Bioluminescence resonance energy transfer. In The Encyclopedia of Genetics, Genomics,  Proteomics and Bioinformatics, ISBN : 0-470-84974-6, J.B. Jorde, P.F.R. Little, M.J. Dunn and S. Subramaniam, Eds., John Wiley & Sons, Ltd. (2005).

    15] Jockers R., and Marullo S. : Basic techniques : Bioluminescence resonance energy transfer. In The Encyclopedia of Genetics, Genomics,  Proteomics and Bioinformatics, ISBN : 0-470-84974-6, J.B. Jorde, P.F.R. Little, M.J. Dunn and S. Subramaniam, Eds., John Wiley & Sons, Ltd. (2005).

    16] Jockers R., Kamal M., Felletschin B. : Comparison of filter sets for BRET1 assays: ß-arrestin2 (ßARR2) recruitment to the vasopressin V2 receptor.
    (Application Note: http://www.berthold.com/ww/en/pub/bioanalytik/overview/notes.cfm).

     17] Achour L, Kamal M, Jockers R and Marullo S: Using quantitative BRET to assess GPCR homo- and hetero-dimerization. In Methods Mol Biol, 756:183-200, series editor; Signal Transduction Protocols. Eds : LM Luttrell. Human Press Inc Totowa, NJ, Vol 83, (2011).

    18] Bouvier M, Heveker N, Jockers R, Marullo S, Milligan G: BRET analysis of GPCR oligomerization: newer does not mean better. Nat Methods, 2007, 4:3-4