Abstracts Biological Sciences

Optimizing fluorescence resonance energy transfer measurements

by Ákos Fábián

Fluorescence resonance energy transfer (FRET) is a non-radiative short distance transfer of energy from an excited donor to an acceptor molecule. The rate of energy transfer is inversely proportional to the sixth power of the distance between the donor and acceptor, therefore the efficiency of energy transfer strongly depends on the intermolecular distance of the donor to the acceptor. This distance dependence was exploited by Stryer and Haugland who first utilized FRET as a “spectroscopic ruler” to measure the distance between two molecules. FRET theory describes the interaction of one donor with one acceptor, however in biological samples interaction of one donor with one acceptor is not guaranteed. Multiple acceptors interacting simultaneously with the same donor will increase the rate constant of energy transfer. This results in increased transfer efficiency without closer proximity between donor and acceptor. We therefore sought to investigate how measured transfer efficiency is influenced by multiple interacting fluorophores. This was achieved by varying the fluorophore-to-protein ratio of fluorescently labeled antibodies and observing the changes in FRET efficiency in an intramolecular model system. Originally, FRET was limited to viewing the interaction between one donor and one acceptor species. However, in the early 2000’s it was realized that the addition of a third dye could expand the capabilities of traditional FRET measurements. First of all, functioning as a relay point, the third dye increased the interaction range that could be viewed with FRET. Secondly, the third dye allowed FRET to be viewed between three distinct molecular species, so that the relative orientation of three molecules could be assessed at the same time. This has significant potential for biological investigations, where higher order multimers and multi-component signaling complexes play important roles in governing biological function. Several methods have been developed to measure FRET in a three-dye system. However, the complexity of the three dye system required simplification, either through extensive sample preparation, restricted sample selection or neglecting of transfer routes. We therefore developed a new method – tripleFRET – that can be implemented with a broad range of biological samples and does not require specific sample preparation beyond fluorescent labeling. A fluoreszcencia rezonancia energiatranszfer (FRET) egy rövid hatósugarú, sugárzásmentes energia átadás egy gerjesztett donor és egy akceptor molekula között. Az energiatranszfer átmenet valószínűsége fordítottan arányos a donor és akceptor közötti távolság hatodik hatványával, így az energiatranszfer hatékonysága is nagyfokú távolság függést mutat. Ezt a távolság függést használta ki Stryer és Haugland, akik először alkalmazták a FRET-et mint „spektroszkópiai vonalzót”. A FRET elmélete egy donor kölcsönhatását írja le egy akceptorral, azonban biológiai mintákban az egy donor-egy akceptor kölcsönhatás nem biztosítható. Ugyanazzal a donorral egyidejűleg…