Mean centering of ratio spectra for estimating of Förster resonance energy transfer (FRET) profile

In general, FRET refers to situations where the donor and acceptor proteins (or fluorophores ) are of two different types. In many biological situations, however, researchers might need to examine the interactions between two or more proteins of the same type or indeed the same protein with itself, for example if the protein folds or forms part of a polymer chain of proteins or for other questions of quantification in biological cells. Obviously, spectral differences will not be the tool used to detect and measure FRET, as both the acceptor and donor proteins emit light with the same wavelengths and this leads to rank-deficiency in measured data [1-3]. There are several methods for measuring the FRET efficiency by monitoring changes in the fluorescence emitted by the donor or the acceptor with the aim of revealing the conformational changes in protein hybridizations. In this work a new simple solution for accurate extraction of FRET profile and consequently determination of FRET efficiency is proposed. Mean centering of ratio spectra [4, 5] was used in two directions on unfolded excitation-emission data matrix obtained for different concentrations of components for eliminating the donor and acceptor pure contributions. The accurate profile related to hybrid component making FRET phenomenon retained. The method was applied on several simulated data matrices in the presence of noise with different degrees of spectral overlapping to evaluate its efficiency toward extraction of true FRET profiles. The effect of unknown interferent on the obtained responses was also tested.