BRET – Bioluminescence Resonance Energy Transfer
BRET (Bioluminescence Resonance Energy Transfer) can be measured in microplate readers to study molecular interactions, especially in the field of G-protein coupled receptor research. BRET is based on the fact that the energy derived from a luciferase reaction can be used to excite a fluorescent protein if the latter is in close proximity to the luciferase enzyme. The technology involves fusion of donor (luciferase) and acceptor (fluorescent) molecules to proteins of interest. Co-expression of fusion constructs in living cells enables their interaction to be studied in real time in a quantitative manner. Energy is transferred from the donor to the acceptor when in close proximity (typically, within 10 nm), resulting in fluorescence emission at a characteristic wavelength. The energy emitted by the acceptor relative to that emitted by the donor is termed the BRET signal, or BRET ratio; it is dependent upon the spectral properties, ratio, distance and relative orientation of the donor and acceptor molecules, as well as the strength and stability of the interaction between the proteins of interest. The ability to study interactions in living mammalian cells circumvents many of the problems associated with techniques such as co-immunoprecipitation and yeast two-hybrid screening. Furthermore, the high sensitivity of BRET enables the study of proteins at physiological concentrations, a significant advantage over techniques that require high levels of protein expression.
As BRET does not require external light source to excite the donor, it has very low background and does not suffer from issues often associated with FRET, such as autofluorescence, light scattering, or photobleaching. Other advantages of BRET are that it is a non-radioactive and homogeneous technology, and that the ratiometric signal minimises interferences from assay conditions. The measurement of BRET requires the use of a luminescence reader equipped with filters; hence multimode readers are often used.
Especially in the field of G-protein coupled receptor research, BRET technology offers the opportunity to establish a homogeneous, universal and functional assay, taking advantage of the fact that ß-arrestin (which is naturally playing a role in the desensitation of the receptors) binds to the intracellular part of the activated receptor. G-protein coupled receptors, also referred to as 7-transmembrane (7TM) receptors, comprise the largest and most diverse superfamily of proteins known. To a minor part only the ligands are known.
To get some idea of how important GPCRs are in drug discovery:
- Currently ~ 30 % of drugs are targeted against GPCRs
- Only 5 % of the known receptors are targeted with drugs
- To only 20 % of the rest the corresponding ligands are known
Over the last years different BRET methods have been developed. All of them have their limitations and benefits. Various donor and acceptor pairs and their corresponding wavelengths can be found in the table below:
|Method||Donor||Substrate||Donor Emission [nm]||Acceptor||Acceptor Emission [nm]|
|BRET 2||RLuc||Deep Blue C™||395||GFP||510|
|eBRET 2||RLuc8||Deep Blue C™||395||GFP||510|
The original BRET method using Coelenterazine as substrate is nowadays called BRET 1. It is characterized by strong signals and long life-time.
In comparison with BRET 1, it has better separated donor and acceptor emission peaks. This makes BRET 2 a better choice for screening assays where high signal to noise ratios are required. A clear limitation of BRET 2 is the low light emission and the short life-time.
Enhanced BRET 2 (eBRET)
eBRET leads to approximately 5-fold better signal as in the original BRET 2 version. eBRET uses a new Renilla luciferase mutant, Rluc8.
The Firefly luciferase in BRET 3 shows lower cellular autofluorescence at the emission wavelength (565 nm) but disadavantages are weak signals and overlap between donor and acceptor emission peaks.
A brand new BRET version is the Quantum Dot-BRET (QD-BRET). The emission peaks are clearly separated which makes QD-BRET ideal for screening applications. Disadvantages are the large size of the QD molecules (1.5 - 6 nm) and the fact that genetical coding of QD-proteins is not possible. QD proteins cannot be expressed in living cells but must be added.
Literature: Bacart et al. (2008): The BRET technology and its application to screening assays, Biotechnology Journal 2008, 3, 311–324