Time-Resolved Förster Resonance Energy Transfer-Based Technologies to Investigate G Protein-Coupled Receptor Machinery: High-Throughput Screening Assays and Future Development - Progress in Molecular Biology and Translational Science - page 278

the HTS campaigns. The principle of FRET and TR-FRET will be only
briefly described here. For more comprehensive explanations, readers
may refer to specific reviews. 3,11,12

2.1. Principle of FRET
FRET is a nonradiative transfer of energy between two fluorophores, a do-
nor and an acceptor, and depends on three conditions: First, the donor emis-
sion spectrum must partially overlap the acceptor excitation spectrum to
allow a transfer of energy; second, the two fluorophores must be in close
proximity, usually at a distance of less than 100 ˚ ; and, third, the orientations
of the fluorophore dipole moments must not be perpendicular to each other
(otherwise no energy transfer can occur). The efficacy of energy transfer ( E )
that is measured is inversely proportional to the sixth power of the distance
between the two chromophores ( R ):

1

E ¼

1 þ

R 6
R 0 6
where R 0 is the F¨ rster radius for a given fluorophore couple. This is a con-
stant depending on the spectral properties of the dyes as well as their relative
orientation and is defined as the distance at which E

50%, such that it can
be calculated or determined experimentally. Because of the distance depen-
dence, FRET measurement is an ideal technique to follow movements
occurring at the level of proteins, in the nanometer range, and it is the reason
why Stryer and colleagues defined it as a “spectroscopic ruler.” 13 Depending
on the position of the dyes on the protein, it can be used to analyze confor-
mational changes within a single protein (e.g., one GPCR) or to monitor
protein-protein interactions (e.g., association between two GPCRs). His-
torically, genetically encoded fluorescent proteins such as CFP and YFP
were fused to the protein of interest, but nowadays, because of their size,
genetically encoded fluorescent proteins are often replaced by small organic
dyes that can be introduced at more precise locations in the protein. An
overview of some techniques available to label proteins with FRET-
compatible organic fluorophores will be presented in Chapter 4.

¼

2.2. Principle of TR-FRET
Just like classic FRET, TR-FRET consists of a nonradiative energy transfer
from a donor molecule to an acceptor fluorophore, but it is based on the use
of particular donors: lanthanide ions (also called rare earths). The peculiarity