Photoswitching FRET to monitor protein–protein interactions

Significance Since protein–protein interactions are extremely important in understanding normal and abnormal cell behavior, cell biologists have often relied on fluorescence techniques, such FRET, as a way to monitor interactions. FRET occurs when donor fluorescent molecules tagged to a protein of interest transfer their excited-state energy to acceptor molecules tagged to another protein of interest. The need to accurately and easily measure FRET led to our development of photoswitching FRET (psFRET). It provides advantages normally associated with advanced methods, like fluorescence lifetime imaging microscopy, with the ease and accessibility of other widely used methods, such as sensitized emission or acceptor photobleaching. The psFRET approach will greatly enhance the ability of cell biologists to utilize FRET in their studies. FRET is a powerful approach to study the interactions of fluorescent molecules, and numerous methods have been developed to measure FRET in cells. Here, we present a method based on a donor molecule’s photoswitching properties, which are slower in the presence vs. the absence of an acceptor. The technique, photoswitching FRET (psFRET), is similar to an established but underutilized method called photobleaching FRET (pbFRET), with the major difference being that the molecules are switched “off” rather than photobleached. The psFRET technique has some of the FRET imaging advantages normally attributed to fluorescence lifetime imaging microscopy (FLIM), such as monitoring only donor fluorescence. However, it can be performed on a conventional widefield microscope, requires less illumination light to photoswitch off than photobleaching, and can be photoswitched “on” again to repeat the experiment. We present data testing the validity of the psFRET approach to quantify FRET in cells and demonstrate its use in imaging protein–protein interactions and fluorescent protein-based biosensors.