New rule(r)s for FRET.

In this issue of the Biophysical Journal, Jarecki et al. (1) investigate the possibility of combining two strategies commonly used to measure distances between loci in a number of systems. The first strategy, Förster resonance energy transfer (FRET), is a spectroscopic method based on energy transfer between two fluorescent molecules: upon transferring its excitation energy to an acceptor via a long-range dipoledipole coupling mechanism, the donor molecule returns to a ground state (2,3). Because this energy transfer can be measured and is inversely proportional to the sixth power of the distance between both fluorophores, the FRET technique lends itself to determining small interor intramolecular distances in vitro or in living cells (3). As a result, FRET has been a key technology in revealing the molecular dynamics underlying various biological phenomena (2). However, the rather large size of commonly used genetically encoded fluorophore probes, their relative orientation, and the need for flexible linkers that occupy a substantial conformational space, complicate accurate distance measurements below ~20 Å (2,3). Nonetheless, experiments to improve FRET distance estimates by incorporating transition metal-ion binding sites within proteins or employing small cysteine-reactive organic fluorophores such as bimane, have shown great promise (4). The second strategy to examine protein dynamics on a nanometer scale in-