Abstract
Bioluminescence resonance energy transfer (BRET) seems to be a promising biophysical technique to study protein–protein interactions within living cells due to a very specific reaction of bioluminescence that essentially decreases the background of other cellular components and light-induced destruction of biomacromolecules. An important direction of the development of this technique is the study of known strong protein–protein complexes in vivo and the estimation of an average distance between chromophores of the donor and acceptor. Here, we demonstrate an in vivo interaction between barnase fused with luciferase (from Renilla reniformis, RLuc) and barstar fused with EGFP (enhanced green fluorescent protein of Aequorea victoria) monitored by BRET. The distance between the luciferase and EGFP chromophores within the complex has been evaluated as equal to (56 ± 2) Å.
Highlights
Allows Monitoring the Bioluminescence resonance energy transfer (BRET) is widely distributed in nature and provides color-shifted light emission of a number of marine and other organisms in the absence of an external light source [1,2]
In contrast to fluorescence resonance energy transfer (FRET), the bioluminescence emission is produced as a result of oxidation of the luciferase cofactor [9,10], which allows using BRET in the presence of intensive scattering, autofluorescence, and photobleaching
BRET has an important advantage over FRET, i.e., the possibility to shift the equilibrium toward complex stabilization through increasing excess of the GFP-fused partner, because its fluorescence background is absent in the case of BRET
Summary
Allows Monitoring the Bioluminescence resonance energy transfer (BRET) is widely distributed in nature and provides color-shifted light emission of a number of marine and other organisms in the absence of an external light source [1,2]. This phenomenon takes place due to the proximity of two photo-proteins: luciferase (as the donor of bioluminescence) and some fluorescent protein (for example, GFP (green fluorescent protein) as the acceptor of bioluminescence) [3,4]. BRET has an important advantage over FRET, i.e., the possibility to shift the equilibrium toward complex stabilization through increasing excess of the GFP-fused partner, because its fluorescence background is absent in the case of BRET
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