Dynamic Estimation of FRET Correction Factors to Study Redox Protein Interactions

Abstract

Fluorescence resonance energy transfer (FRET) method is a key tool to determine molecular proximity and conformation changes of biomolecules in living cells. As the distance change between donor and acceptor fluorescent proteins attached to target proteins occurs in the range of 1-10 nm, an appreciable energy transfer can be detected at spatial and temporal resolution. Therefore it provides the most sensitive approach to study intercellular protein dynamics. Despite the earlier methods for characterization of FRET efficiency that accounts for donor and acceptor expression levels in cells; spectral bleed-through due to broad emission of fluorescent proteins results in inadequate computation of FRET, therefore efficiency and distance change cannot be determined accurately. Normalized FRET (nFRET) by using correction factors, were used to extract after FRET pair establishes an equilibrium state. However, photobleaching, varying expression of FRET pair, uncorrelated dipole orientations of fluorescent proteins, heterogeneous distance changes of linkers between donor and acceptor result in incomplete computation of nFRET since correction factors were not dynamically determined during data acquisition. Thus, the use of single bleed through coefficient for computation of netFRET values may also results in overestimation of efficiency and distance. We demonstrated that donor and acceptor bleed-through values changes during the data acquisition. Therefore, netFRET cannot be computed by a single donor-acceptor correction factors. Alternatively, we presented here dynamic coefficient estimation method to determine correction factors by using the ratio of emissions from FRET channel and donor/acceptor channels for each frame after fitting pixel intensity values and later used them to compute nFRET efficiencies. The method yields a 0.38 nFRET values for Cerulean-Venus (Cer17Ven) FRET pair separated by a short linker, however the use of static coefficients of average or minimum factors yields 0.34 nFRET value. Single coefficient incorrectly reports distance change when the measurements were taken before an equilibrium state. Furthermore, method was used to measure heme induced intercellular conformation changes of Cytochrome c labeled with Cer and Ven fluorescent pairs. The distance after Cyt c folded by Heme Lyase was accurately computed. As a result, a new approach was demonstrated to determine the nFRET efficiency and its direct use to compute distance changes. Taking all values during the data acquisition eliminated the coefficient dependent variation.