Biophys. J. 105, 2724–2732 (2013)

Credit: BARON CHANDA

Techniques such as Förster resonance energy transfer (FRET) and chemical crosslinking have been used successfully to monitor molecular distances, but these can be limited by considerations such as the distances that can be measured (∼10–100 Å in FRET) and the physical and dynamic properties of the fluorescent groups used. Jarecki et al. developed a strategy that combines the concepts of these two approaches in which their limitations can be mitigated by each other. The authors generated “tethered quenchers” where the short-range quencher nitroxide radical on one end of a molecule quenches a fluorophore on another end by collisional quenching. They first calibrated the approach by generating a series of probes with varied lengths (PEG units) between the quencher and fluorophore on different lengths of polyproline, which are known to form rigid helices of fixed lengths. The authors ultimately determined that the features of quenching report on the length of the quencher, thereby providing a ruler for studying the distances between two unknown molecular points in the range of 4–30 Å. Molecular dynamics simulations provided further validation. Next, the authors used an environmentally sensitive fluorophore in their quencher to read out the Shaker K+ channel's activation state and provide support for the 'resting-state' model where the transitions between channel opening and closing are asymmetric. 'Tethered quenchers' should find utility in even more complex systems such as large protein complexes.