Bioluminescence resonance energy transfer

Green fluorescent protein is commonly used for energy-transfer experiments (Baubet etal. 2000). The fluorescent moiety of GFP protein is the Ser-Tyr-Gly derived chromophore. GFP can be expressed in a variety of cells where it becomes fluorescent, can be fused to a host 

the Phe-64 - Leu and Ser-65 — Thr mutant shows higher fluorescence parameters than that of the wild GFP. Excitation at 458 nm yields a fluorescence spectrum with two peaks at 512 and 530 nm. The fluorescence properties of this enhanced green fluorescent protein (EGFP) were found to be similar to the recombinant glutathione S-transferase-EGFP (GST-EGFP) protein, expressed in Escherichia coli (Cinelli etal. 2004). 

In most BRET applications, the fused donor is RewHaluciferase (Rluc) rather than aequorin, to avoid any intrinsic affinity for Aequorea-derived GFP mutant the acceptor is the YFP, to increase the spectral distinction between the two emissions. When the donor and acceptor are in close proximity, the energy resulting from catalytic degradation of the coelenterazine derivative substrate is transferred from the luciferase to the YFP, which will then emit fluorescence at its characteristic wavelength (Xu etal. 1999). 

Resonance energy transfer is obvious for the positive control. No BRET occurs for the negative control. Courtesy of BMC Labtech, Germany. 

Artificial fusion construct of the positive control (Rluc-GFP2) induces an extremely high BRET. Also, the large spectral resolution between donor and emission peaks in BRET (115 nm) (Angers et al. 2000) greatly improves the signal to background ratio over 

Like FRET, bioluminescence resonance energy transfer (BRET) is based on non-radiative energy transfer between a donor and an acceptor. However, in BRET, the donor is a luminescent molecule, excited by the enzyme Renilla Luciferase (Rluc), and not a fluorescent molecule. The BRET acceptor can be a fluorescent protein like green fluorescent protein (GFP) or YFR If the enzyme and the fluorescent protein are in close proximity (d 10 nm), an energy transfer will occur between the Rluc substrate (coelanterazine) and the fluorescent protein, leading to emission from the later. 

Since Rluc and GEP recombinantly fused proteins can be expressed in living cells, BRET is an interesting tool for monitoring molecular interactions in cell-based assays. BRET has been particularly used for the study of GPCRs by probing receptor oligomerization or activation.  

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Percherancier Y, Berchiche YA, Slight 1, Volkmer-Engert R, Tamamura H, Fujii N, Bouvier M, Heveker N (2005) Bioluminescence resonance energy transfer reveals hgand-induced conformational changes in CXCR4 homo- and heterodimers. J Biol Chem 280 9895-9903... 

James, J. R., Oliveira, M. I., Carmo, A. M., Iaboni, A. and Davis, S. J. (2006). A rigorous experimental framework for detecting protein oligomerization using bioluminescence resonance energy transfer. Nat. Methods 3, 1001-6. 

Coulon, V., Audet, M., Homburger, V., Bockaert, J., Fagni, L., Bouvier, M. and Perroy, J. (2008). Subcellular imaging of dynamic protein interactions by bioluminescence resonance energy transfer. Biophys. J. 94,1001-9. 

Xu, X., Soutto, M., Xie, Q., Servick, S., Subramanian, C., von Arnim, A. G. and Johnson, C. H. (2007). Imaging protein interactions with bioluminescence resonance energy transfer (BRET) in plant and mammalian cells and tissues. Proc. Natl. Acad. Sci. USA 104, 10264-9. 

Cheng, Z. J. and Miller, L. J. (2001) Agonist-dependent dissociation of oligomeric complexes of G protein-coupled cholecystokinin receptors demonstrated in living cells using bioluminescence resonance energy transfer. J. Biol. Chem. 276,48040-48047. 

Mercier, J. F., Salahpour, A., Angers, S., Breit, A., and Bouvier, M. (2002) Quantitative assessment of beta 1 and beta 2-adrenergic receptor homo and hetero-dimerization by bioluminescence resonance energy transfer. J. Biol. Chem. 277,44925 14931. 

Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc. Natl. Acad. Sci. USA 97, 3684-3689. 

Yamakawa Y, Ueda H, Kitayama A et al (2002) Rapid homogeneous immunoassay of peptides based on bioluminescence resonance energy transfer from firefly luciferase. J Biosci Bioeng 93 537-542... 

Alvarez-Curto E, Pediani JD, Milligan G (2010) Applications of fluorescence and bioluminescence resonance energy transfer to drug discovery at G protein coupled receptors. Anal Bioanal Chem 398 167-180... 

Xu, Y., Piston, D.W. and Johnson, C.H. (1999). A bioluminescence resonance energy transfer (BRET) system application to interacting circadian clock proteins. Proceedings of the National Academy of Sciences, USA, 96, 151-156. 

McVey M, Ramsay D, Kellett E, Rees S, Wilson S, et al. 2001. Monitoring receptor oligomerization using time-resolved fluorescence resonance energy transfer and bioluminescence resonance energy transfer. The human delta-opioid receptor displays constitutive oligomerization at the cell... 

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