Bioluminescence Resonance Energy Transfer (BRET) is a scientific technique used to investigate how molecules interact within living cells. This method provides insights into biological processes as they unfold in real-time, offering insights into dynamic cellular functions. By observing these molecular partnerships, researchers can gain a deeper understanding of how biological systems operate and respond to various conditions. BRET has become a valuable tool across various fields of biological research due to its ability to detect interactions in a natural cellular environment.
The Core Principle of BRET
The foundation of BRET lies in two distinct phenomena: bioluminescence and resonance energy transfer. Bioluminescence refers to the light produced by living organisms, a process often seen in creatures like fireflies or certain marine organisms. This light is generated through a chemical reaction involving an enzyme, typically a luciferase, acting upon a specific molecule called a substrate, such as coelenterazine.
BRET also incorporates the principle of resonance energy transfer. This is a non-radiative process where energy moves from a “donor” molecule to an “acceptor” molecule without the donor actually emitting light. This energy transfer can only occur if the donor and acceptor molecules are extremely close to each other, generally within 10 nanometers, and their light absorption and emission properties overlap.
In a BRET assay, a bioluminescent enzyme, usually a luciferase like Renilla luciferase (RLuc) or its brighter variant RLuc8, serves as the donor. This donor is paired with a fluorescent protein, such as Green Fluorescent Protein (GFP) or Yellow Fluorescent Protein (YFP), which acts as the acceptor. When the donor and acceptor are brought into very close proximity, often due to a specific protein-protein interaction, the energy from the bioluminescent reaction of the donor is efficiently transferred to the fluorescent acceptor. This energy transfer causes the fluorescent acceptor to emit light, providing a detectable signal that indicates the interaction.
How BRET Assays Are Performed
Performing a BRET assay begins with preparing the proteins of interest. Each protein is genetically engineered to be “tagged” or fused with either a bioluminescent donor, like luciferase, or a fluorescent acceptor protein, such as GFP or YFP. For instance, if researchers want to study the interaction between two proteins, Protein A and Protein B, Protein A might be fused to luciferase, and Protein B to GFP.
These tagged proteins are then introduced into living cells. Human embryonic kidney (HEK293T) cells are commonly used for this purpose due to their ease of handling and high transfection efficiency. After the cells have expressed both the donor- and acceptor-tagged proteins, the assay is initiated by adding the specific substrate for the luciferase enzyme, such as coelenterazine.
The luciferase enzyme, now active, begins to oxidize its substrate, producing bioluminescence. If the two proteins of interest are interacting and thus bringing the donor and acceptor into close enough proximity (typically less than 10 nanometers), the energy from the donor’s bioluminescent reaction is transferred to the fluorescent acceptor. This energy transfer then excites the acceptor, causing it to emit its own light, which is then measured using specialized equipment, often a microplate reader. The light emitted from the acceptor is differentiated from the light directly emitted by the donor, and a BRET ratio is calculated. A higher ratio indicates a stronger or more frequent interaction between the proteins in the living cell.
Key Applications of BRET Assays
BRET assays are widely used in scientific research to understand various biological mechanisms, particularly for investigating protein-protein interactions. For example, they are frequently employed to study how G protein-coupled receptors (GPCRs), a large family of cell surface receptors, interact with each other in processes like receptor dimerization or with other signaling molecules such as beta-arrestins. This capability allows researchers to observe these dynamic interactions as they happen within a living cellular environment.
Beyond protein interactions, BRET assays are valuable in drug discovery. They can be used to screen large libraries of compounds to identify potential drug candidates that either promote or disrupt specific molecular interactions, which is particularly useful for developing new therapies targeting disease pathways. Performing these screens in live cells provides a more physiologically relevant context for evaluating drug effects.
Furthermore, BRET assays contribute to understanding complex cellular processes like signal transduction, where cells receive and respond to external stimuli. They can also monitor enzyme activity, track protein movement within a cell (protein trafficking), and detect changes in intracellular signaling molecules such as cyclic AMP (cAMP) or calcium ions. BRET provides real-time data from intact cells, offering a comprehensive view of dynamic biological events that might be missed by methods requiring cell disruption.