Protein-Interaction Experiments by Förster Resonance Energy Transfer (FRET)
Förster resonance energy transfer (FRET) is an interaction of two molecules in which the emission band of one molecule overlaps the absorption band of the other. In this case the energy from the first molecule, the donor, can transfer into the second one, the acceptor. FRET can result in an extremely efficient quenching of the donor fluorescence and, consequently, in a considerable decrease of the donor lifetime. The energy transfer rate from the donor to the acceptor decreases with the sixth power of the distance. Therefore it is noticeable only at distances shorter than 10 nm . FRET is used as a tool to investigate protein-protein interaction. Different proteins are labelled with the donor and the acceptor, and FRET is used as an indicator of the binding between these proteins. FRET is the most frequent FLIM application.
Because of its dependence on the distance FRET has become an important tool of cell biology. Different proteins are labelled with the donor and the acceptor; FRET is then used to verify whether the proteins are physically linked and to determine distances on the nm scale.
Single-exponential interpretation of FLIM-FRET data
The interpretation of FLIM-FRET data is simple: In the absence of FRET the lifetime of the donor is unchanged. When FRET is present the donor is loosing its excitation energy into the acceptor, and the lifetime decreases, see figure below.
The use of FLIM for FRET has the obvious benefit that the FRET intensity is obtained from a single lifetime image of the donor. When FRET occurs the fluorescence lifetime of the donor decreases, when FRET is absent it remains constant. All that is needed to detect protein interaction is a single lifetime image of the donor. Donor bleedthrough and directly excited acceptor fluorescence therefore have no influence of FLIM-FRET measurements. An example is shown in the figure below.
In all protein-interaction experiments, there is usually a mixture of interacting and non-interacting proteins. Both the fraction of interacting proteins and the distance between the proteins influence the net FRET efficiency derived from the intensities. It therefore cannot be told whether a variation in FRET efficiency is due to a variation in the distance or a variation in the fraction of interacting proteins.
TCSPC FLIM solves the problem of interacting and non-interacting donor by double-exponential lifetime analysis. The resulting donor decay functions can be approximated by a double exponential model, with a fast component from the interacting donor molecules and a slow lifetime component from the non-interacting donor molecules. There are several reasons why a donor does not interact. The protein may just not be linked to each other, an acceptor protein may not be labelled with the acceptor, or the orientation between the donor and the acceptor may be wrong. Orientation is usually considered random, and taken into account by the κ2 factor. If the labelling is complete, as it can be expected if the cell is expressing fusion proteins of the GFP variants, the decay components represent the fractions of interacting and non-interacting donor molecules. Corrected by κ2, the amplitudes, a and b, then represent the fractions of interacting and non-interacting protein molecules. The composition of the donor decay function is illustrated in the figure below.
The next figure shows a result of a double-exponential FLIM-FRET analysis. The left image shows the ratio of the lifetimes of the non-interacting and interacting donor fractions, t0/tfret. The distribution of t0/tfret in different regions is shown far left. The locations of the maxima differ by only 10 %, corresponding to a distance variation of about 2 %. However, the variation in the intensity coefficients, a/b, and thus in the ratio of interacting proteins, Nfret/N0 ,is about 10:1.
The result shows clearly that the variation in the single-exponential lifetime is almost entirely caused by a variation in the fraction of interacting proteins, not by a change in donor-acceptor distance. In other words, interpreting variations in the single-exponential lifetime (or classic FRET efficiencies from steady-state experiments!) as distance variations leads to wrong conclusions. Double-exponential FRET analysis is therefore a substantial step towards quantitative FRET experiments.
For more information and references please see bh TCSPC Handbook, chapter Förster Resonance Energy Transfer (FRET).
References for FRET Imaging
For more references on FLIM-FRET please see bh TCSPC Handbook, chapter ‘Förster Resonance Energy Transfer (FRET)’.
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