Should we fret about FRET?

21 June 2013

Restricted State Selection in Fluorescent Protein Förster Resonance Energy Transfer

Thomas A. Masters, Richard J. Marsh, Daven A. Armoogum, Nick Nicolaou, Banafshé Larijani, Angus J. Bain

Journal of the American Chemical Society, 2013, 135 (21), pp 7883–7890

When certain molecules get close together, a few nanometers apart, energy can be transferred between them. If one molecule is electronically excited, this energy can be transferred to the other by a process known as Förster resonance energy transfer (FRET). FRET has been referred to as the “nanometre ruler” and allows the measurement of intermolecular distances and conformational change without recourse to invasive techniques such as electron microscopy.

With the development of fluorescent proteins derived from the Aequorea victoria jellyfish, it has been possible to induce cells to make these fluorescent proteins themselves and to attach them to molecules of interest within the cell itself. This has revolutionised the bio-sciences and led to the award of the Nobel Prize in Chemistry in 2008.

FRET using fluorescent proteins is one of the main tools for investigating cell biophysics and cell biochemistry, two of the most widely used proteins are the enhanced green fluorescent protein (EGFP) as the donor and the red shifted variant mCherry as the FRET acceptor. However it has been noticed that in some cases, FRET does not work as efficiently as it should. In a recent collaboration between UCL researchers and Banafshe Larijani’s group at Cancer Research UK’s London Research Institute, it has been shown that two electronic states in EGFP and mCherry should be involved in FRET, but in fact only one state in each protein takes part.

Their work uses the novel combination of fluorescence lifetime and polarisation measurements to reveal this surprising discovery and the results indicate that a number of established FRET techniques would not detect such restrictions. Fretting about FRET is lessened by the time resolved spectroscopic techniques they have developed. This research was supported by Cancer Research UK, EPSRC and UCL Enterprise.

Restricted State Selection in Fluorescent Protein Förster Resonance Energy Transfer