Bioluminescence Resonance Energy Transfer (BRET) is a research method for observing molecular interactions inside living cells. It combines two phenomena: bioluminescence and resonance energy transfer. Bioluminescence is when a living organism produces light through a chemical reaction, like a firefly. Resonance energy transfer is when one excited molecule passes its energy to a neighbor without emitting light.
The BRET technique uses these principles to determine if molecules, such as proteins, are physically close enough to be interacting. By attaching light-producing and light-receiving tags to different molecules, scientists can effectively “see” when these molecules come together. This provides a window into the dynamic processes occurring within a cell.
The BRET Mechanism
The mechanism of BRET involves a “donor” and an “acceptor” molecule. The donor is a bioluminescent enzyme, like Renilla luciferase from a sea pansy. The acceptor is a fluorescent protein, like Yellow Fluorescent Protein (YFP). Scientists genetically fuse the donor to one molecule of interest and the acceptor to a second molecule.
When these molecules are in a cell, the process starts by adding a substrate like coelenterazine. The luciferase donor uses this substrate to generate light, usually blue, in an enzymatic reaction.
If the molecules are not interacting, the donor’s blue light is the only signal detected. If the molecules are in close proximity—within 1 to 10 nanometers—the energy from the donor is transferred directly to the nearby acceptor protein in a non-radiative process.
This energy transfer excites the acceptor protein, causing it to emit its own light in a distinct color, such as yellow, which is the BRET signal. Scientists measure the ratio of the acceptor’s light to the donor’s light to quantify the interaction. A high ratio indicates the molecules are interacting.
Key Applications in Research
One widespread application of BRET is studying protein-protein interactions. Many cellular functions are carried out by proteins working together in complexes, and BRET allows researchers to confirm these partnerships. This provides direct evidence of functional relationships that might otherwise be inferred.
BRET is also used to analyze the activity of cell surface receptors, which is relevant for drug development. It can monitor how a receptor interacts with other proteins after being activated by a hormone or a potential drug. This allows researchers to screen compounds to see which ones turn a specific receptor on or off, helping identify therapeutic candidates.
The technique can also detect conformational changes, which are subtle shifts in a single protein’s shape. These changes often act as a molecular switch for a protein’s function. By placing the BRET donor and acceptor on different parts of the same protein, scientists can see if it folds or unfolds in response to a specific cellular event.
Comparing BRET and FRET
BRET is often compared to a similar technique, Förster Resonance Energy Transfer (FRET). Both measure molecular proximity, but their primary difference is the donor’s energy source. FRET requires an external light source, such as a laser, to excite its donor. In contrast, BRET’s donor generates its own light through a chemical reaction with a substrate.
This distinction leads to practical advantages for BRET. The external light used in FRET can damage living cells (phototoxicity) or cause the fluorescent molecules to lose their ability to emit light (photobleaching). BRET avoids these issues because it does not use an external light source, making it a gentler method for longer studies.
BRET also produces a cleaner signal. The external excitation light in FRET can cause background fluorescence from other molecules in the cell, creating noise that can obscure the signal. Since BRET’s light is generated internally, it eliminates this background noise, resulting in a higher signal-to-noise ratio.
Evolution of BRET Technologies
The BRET technique has been refined over the years. The first versions, BRET1 and BRET2, used Renilla luciferase paired with a Yellow Fluorescent Protein. These early versions had limitations, including low light output and spectral overlap, where the donor and acceptor light colors were too similar to distinguish clearly.
BRET2 was developed to address spectral overlap by using a different substrate, DeepBlueC™, which shifted the donor’s light emission. This created a larger separation between the donor and acceptor signals, improving measurement accuracy. However, BRET2 systems produced a weaker signal and had a faster signal decay compared to BRET1, limiting their application.
NanoBRET is a newer generation that offers improvements. This system uses NanoLuc, a much smaller and brighter luciferase enzyme engineered from a deep-sea shrimp. Its intense light emission increases the sensitivity of the assay, allowing researchers to study proteins at lower, more natural expression levels.
The small size of the NanoLuc donor is another benefit, as it is less likely to interfere with the normal function of the protein it is attached to. This ensures that the observed interactions are more representative of the biological process. These enhancements have made NanoBRET a widely adopted tool for studying molecular dynamics.