Abstract
Protein–protein interaction studies often provide new insights, i.e., into the formation of protein complexes relevant for structural oligomerization, regulation of enzymatic activity or information transfer within signal transduction pathways. Mostly, biochemical approaches have been used to study such interactions, but their results are limited to observations from lysed cells. A powerful tool for the non-invasive investigation of protein–protein interactions in the context of living cells is the microscopic analysis of Förster Resonance Energy Transfer (FRET) among fluorescent proteins. Normally, FRET is used to monitor the interaction state of two proteins, but in addition, FRET studies have been used to investigate three or more interacting proteins at the same time. Here we describe a fluorescence microscopy-based method which applies a novel 2-step acceptor photobleaching protocol to discriminate between non-interacting, dimeric interacting and trimeric interacting states within a three-fluorophore setup. For this purpose, intensity- and fluorescence lifetime-related FRET effects were analyzed on representative fluorescent dimeric and trimeric FRET-constructs expressed in the cytosol of HEK293 cells. In particular, by combining FLIM- and intensity-based FRET data acquisition and interpretation, our method allows to distinguish trimeric from different types of dimeric (single-, double- or triple-dimeric) protein–protein interactions of three potential interaction partners in the physiological setting of living cells.
Highlights
Förster or fluorescence resonance energy transfer (FRET) is a physical effect between two interacting fluorophores called the FRET donor and the FRET acceptor (Clegg, 1995; Bajar et al, 2016)
These fluorophores were chosen for the following reasons: 1) The spectral properties of these fluorophores exhibit a clear overlap between the emission spectrum of the FRET donor and the excitation spectrum of the FRET acceptor for every possible interaction (TY, TC, and YC) (Supplementary Figures 1A–C), which is a prerequisite for the final energy transfer
The calculated FRET efficiencies of the TY construct obtained from the amplitudeweighted lifetime (74.6 ± 1.6%) corresponded better to the FRET efficiencies observed by acceptor photobleaching experiments (Supplementary Figure 2B) than the FRET efficiencies calculated from the intensity-weighted lifetime (40.7 ± 2.7%)
Summary
Förster or fluorescence resonance energy transfer (FRET) is a physical effect between two interacting fluorophores called the FRET donor and the FRET acceptor (Clegg, 1995; Bajar et al, 2016). There is sensitized emission of the acceptor while exciting the donor, a phenomenon that is measured by corrected FRET or spectral imaging approaches These approaches have been developed to study FRET between two fluorophores and only provide insights into the interaction of two proteins. Most microscopy studies have focused on detecting sensitized emission of the acceptor to study FRET among multiple fluorophores This approach seems to be advantageous for the imaging of living cells, since much faster acquisition rates can be achieved compared to fluorescence lifetime or acceptor photobleaching set-ups. We report a method to distinguish non-interacting, dimeric and trimeric interacting proteins in living cells using a two-step acceptor photobleaching protocol for a three-fluorophore setup This method provides a snapshot of the state of protein interactions and cannot be used for repeated measurements on the same cell. By using a novel combination of FLIM- and intensity-based FRET data interpretation applied for the same cell, our method can be used to reliably distinguish trimeric from different types of dimeric (single-, double- or triple-dimeric) protein–protein interactions of three potential interaction partners in living cells
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