Improving the spectral analysis of fluorescence resonance energy transfer in live cells: Application to interferon receptors and Janus kinases

Abstract

The observed Fluorescence Resonance Energy Transfer (FRET) between fluorescently labeled proteins varies in cells. To understand how this variation affects our interpretation of how proteins interact in cells, we developed a protocol that mathematically separates donor-independent and donor-dependent excitations of acceptor, determines the electromagnetic interaction of donors and acceptors, and quantifies the efficiency of the interaction of donors and acceptors. By analyzing large populations of cells, we found that misbalanced or insufficient expression of acceptor or donor as well as their inefficient or reversible interaction influenced FRET efficiency in vivo. Use of red-shifted donors and acceptors gave spectra with less endogenous fluorescence but produced lower FRET efficiency, possibly caused by reduced quenching of red-shifted fluorophores in cells. Additionally, cryptic interactions between jellyfish FPs artefactually increased the apparent FRET efficiency. Our protocol can distinguish specific and nonspecific protein interactions even within highly constrained environments as plasma membranes. Overall, accurate FRET estimations in cells or within complex environments can be obtained by a combination of proper data analysis, study of sufficient numbers of cells, and use of properly empirically developed fluorescent proteins.

Ligand-independent interaction of the type I interferon receptor complex is necessary to observe its biological activity

“…Discovering that using non-jellyfish red-shifted truly monomeric fluorescent proteins gives more precise FRET determinations and removes artefacts arising from interactions between jellyfish fluorescent proteins [11], we used fluorescent protein pairs derived from distinct species (e.g., jellyfish-derived monomeric green or citrine fluorescent proteins, EGFP and CiFP, or anemone-derived teal fluorescent protein, TFP, as donors, and coral-derived orange or strawberry fluorescent proteins, OFP and StFP, as acceptors).…”

“…All primers in this manuscript are written in the 5′-3′ orientation. The synthesis of the various cDNA’s used in this manuscript is described in Supplementary Text 1 of this manuscript and elsewhere [11]. …”

“…FRET between fluorescent protein donor:acceptor pairs ECFP and EYFP in intact cells was measured with an instrument in which a spectrometer is integrated into the sidebox of a confocal microscope, as described previously [9;11–13]. …”

“…The improved analysis of deconvolved spectra, calculation of FRET efficiency, other cellular biophysical quantities, and the inter-FP distances are described elsewhere [11]. …”

“…Transfections using DDAB:DOPE liposomes and polyethyleneimine (PEI) were performed as described elsewhere [11;19]. …”