FRET and BRET Techniques

Most of these assays involve resonance energy transfer (RET) techniques. RET is a short-range, nonradiative energy transfer between donor and acceptor molecules that takes place only if the two species are in close proximity (< 10 nm) to each other. RET systems are therefore "spectroscopic rulers" that are suitable for monitoring interactions between two partners, provided that each component of the donor-acceptor couple is linked to one partner. The same strategy can be used to detect protein conformation changes if donor and acceptor are fused at the target protein terminals.

In the case of fluorescence RET (FRET), both the donor and the acceptor are fluorescent molecules, whereas in bioluminescence RET (BRET) a bioluminescent molecule acts as the energy donor. FRET and, more recently, BRET are the methods of choice for imaging protein association inside living cells. In addition, RET processes can be evaluated using microtiter plate readers, thus allowing for the development of FRET- and BRET-based quantitative assays.

FRET methods can be broadly classified as intensity-based and decay kinetics-based methods. Intensity-based methods rely on the measurement of either the acceptor fluorescence or the acceptor:donor fluorescence intensity ratio. However, they are not suitable for detecting low-level protein-protein interactions because their sensitivity is reduced by several factors (e.g., sample autofluorescence and overlap between donor and acceptor emission). Such problems have been partially overcome by other FRET technologies, such as time-resolved FRET (TR-FRET), in which the donor is a lanthanide chelate with long-lived fluorescence. Decay kinetics-based FRET methods rely on the measurement of the kinetics of the light emission. In fluorescence lifetime imaging microscopy (FLIM), the decrease in the donor fluorescence lifetime that is due to FRET is measured, providing a reliable, donor concentration-independent estimation of FRET efficiency. Because the fluorescence lifetimes of donors are in the pico- or nanosecond range, even extremely rapid interactions can be monitored. In addition, unlike intensity-based FRET, FLIM measures can be also performed with spectrally similar fluorophores.

The BRET process occurs naturally in some marine organisms, such as the sea pansy Renilla reniformis and the jellyfish Aequorea victoria. In both cases the green fluorescent protein (GFP) is the acceptor, while Renilla luciferase (Rluc) or aequorin is the donor, respectively. BRET possesses some advantages in comparison with FRET because it avoids the problems associated with illumination. Therefore,

9.2 Protein-Protein and Protein-Ligand Interaction Assays 1159

it can be used even in photoresponsive cells or in cell types with significant autofluorescence. A further advantage of BRET is its higher sensitivity, which allows protein-protein interactions to be measured at low protein concentration, thus reducing nonspecific association phenomena of the target proteins. Because BRET assays are essentially intensity-based assays, spectral separation of donor and acceptor emissions is required.

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Responses

  • bryn
    How fret is different from bret phenomenon?
    2 years ago

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