Fluorogen Activating Peptide Based Energy Transfer Donors for FRET in Living Cells

Abstract

Ensemble FRET based biosensors have been demonstrated by a number of groups that allow for direct interrogation of signaling pathways in living cells, with a range of activities including protein modification, protein conformational changes, and protein interactions. The response strength of these probes is limited by FRET efficiency and ensemble averaging, as the responses typically come from a heterogeneous population of responders in the dynamic cellular situation. We present a novel approach to FRET in living cells that allows single molecule FRET determinations based on stochastic activation of an expressible donor moiety. This approach allows low-density single molecule FRET measurements to be determined in living cells, and correlated to cellular location.We have isolated Fluorogen Activating Peptides (FAPs) based on single chain antibody fragments that noncovalently bind to dyes and enhance their fluorescence by factors of 2000-20,000. In the apo-form, the FAP has no chromophore or detectable fluorescence, while diffusion controlled binding of dye results in “switching” of the module with a very high contrast ratio. Use of FAP-based FRET donors, in low concentrations of fluorogen with respect to the FAP-Fluorogen Kd results in a low occupancy of FRET donors when paired with other expressible FRET acceptors, such as RFP or ReAsH domains. This stochastic occupancy allows single molecule determinations of FRET efficiency, at a rate equal to the object localization rate, governed by binding of the fluorogen to the FAP (see posters by Qi Yan et al. and Keith Lidke et al.), and breaking from the ensemble averaged FRET measurements. We present data and formalisms for analysis of the FRET efficiency of incompletely occupied ensembles of molecules, and feasibility demonstrations for single-molecule FRET determination using sparse single molecule measurements on living cells.