The Reach construct

The FRET (Förster Resonance Energy Transfer) System

Förster resonance energy transfer (FRET), sometimes also called fluorescence resonance energy transfer, is a physical process of energy transfer named after the German physical chemist Theodor Förster. In FRET, the energy of a donor chromophore, whose electrons are in an excited state, is passed to a second chromophore, the acceptor. The energy is transferred without radiation and is therefore not exchanged via emission and absorption of photons. In biochemistry and cell biology, fluorescent dyes, which interact via FRET, are applied as "optical nano metering rules", because the intensity is depend on the spacing between donor and acceptor, and can be observed up to distances of 10 nm. This way, protein-protein interactions and conformational changes of a variety of tagged biomolecules can be observed.

However, fluorescence is not an essential requirement for FRET. This type of energy transfer can also be obersved between donors that are capable of other forms of radiation, such as phosphorescence, bioluminescence or chemiluminescence, and fit acceptors. Acceptor chromophores do not necessarily emit the energy in form of light, and can lead to quenching. Thus, this kind of acceptors are also referred to as dark quenchers. In our project, we use a FRET system with a dark quencher, namely our Reach construct.

REACh proteins - dark quenchers of GFP

In 2006, Ganesan et al. were the first to present a previously undescribed FRET acceptor, a non-fluorescent yellow fluorescent protein (YFP) mutant called REACh (for Resonance Energy-Accepting Chromoprotein). YFP can be used as a FRET acceptor in combination with GFP as the donor in FRET microscopy and miscellaneous assays in molecular biology. The ideal FRET couple should possess a large spectral overlap between donor emission and acceptor absorption but separated emission spectra to allow their selective imaging.

To optimize the spectral overlap of this FRET pair, the group obtained a genetically modified YFP acceptor. Mutations of amino acid residues that stabilize the excited state of the chromophore in enhanced YFP (EYFP) resulted in a non-fluorescent chromoprotein. Two mutations, H148V and Y145W, reduced the fluorescence emission by 82 and 98 %, respectively. Ganesan et al. chose the Y145W mutant and the Y145W/H148V double mutant as FRET acceptors and named them REACh1 and REACh2, respectively. Both REACh1 and REACh2 act as dark quenchers of GFP.

Producing a GFP_Reach Fusion Protein

Cutting the fusion protein with the TEV Protease

A Fluorescence answer faster than expression