Mathematical models for quantitative assessment of bioluminescence resonance energy transfer: application to seven transmembrane receptors oligomerization

Abstract

The idea that seven transmembrane receptors (7TMRs; also designated G-protein coupled receptors, GPCRs) might form dimers or higher order oligomeric complexes was formulated more than 20 years ago and has been intensively studied since then. In the last decade, bioluminescence resonance energy transfer (BRET) has been one of the most frequently used biophysical methods for studying 7TMRs oligomerization. This technique enables monitoring physical interactions between protein partners in living cells fused to donor and acceptor moieties. It relies on non-radiative transfer of energy between donor and acceptor, depending on their intermolecular distance (1-10 nm) and relative orientation. Results derived from BRET-based techniques are very persuasive; however, they need appropriate controls and critical interpretation. To overcome concerns about the specificity of BRET-derived results, a set of experiments has been proposed, including negative control with a non-interacting receptor or protein, BRET dilution, saturation, and competition assays. This article presents the theoretical background behind BRET assays, then outlines mathematical models for quantitative interpretation of BRET saturation and competition assay results, gives examples of their utilization and discusses the possibilities of quantitative analysis of data generated with other RET-based techniques.

Keywords: 7TMRs; BRET; mathematical models; oligomerization; quantitative analysis.

Figure 1

Schematic representation of the BRET assay and various BRET variants for studying protein–protein interaction. (A) BRET enables monitoring of physical interactions between two proteins genetically fused to donor and acceptor molecules. The BRET donor is a bioluminescent enzyme (a version of Renilla luciferase, Rluc), which reacts with the substrate to produce excitation. The acceptor molecule is usually a version of a green fluorescent protein (GFP). If the distance between donor and acceptor is more than 10 nm, light is emitted with an emission spectra characteristic for Rluc. When the distance is less than 10 nm, part of this energy is non-radiatively transferred by RET from donor (Rluc) to acceptor (version of GFP), resulting in an additional signal emitted by the acceptor. (B) A summary of BRET variants and their basic characteristics.

Figure 2

Theoretical BRET dilution curves. The ratio between acceptors and donors is kept constant.

Figure 3

BRET saturation assay. Theoretical curves for oligomer formation are plotted as a function of the ratio of receptors tagged with acceptor [A] and donor [D] molecules. In the case of monomers, the BRET signal is created by random collisions.

Figure 4

Bioluminescence resonance energy transfer saturation curves for dimers with different energy transfer efficiencies E.

Figure 5

Bioluminescence resonance energy transfer competition curves. In a homologous assay, the same kind of receptor is used as a competitor, whereas in a heterologous assay, a different receptor with a smaller affinity for hetero-dimer formation is used.