The Chemistry Department Presents: Novel (and novice) imaging of Forster Resonance Energy Transfer (FRET) using photoswitchable fluorescent proteins

Dr. George Patterson

Monitoring of protein-protein interactions and oligomerization has benefited greatly from Förster Resonance Energy Transfer (FRET) measurements. Work in our lab has been directed toward developing a straightforward FRET techniques, resulting in the development of new methods relying on a unique class of fluorescent proteins called photoswitchable fluorescent proteins. One technique, photoswitching FRET (psFRET), similar to an established, underutilized method called photobleaching FRET (pbFRET) with the major difference being the molecules are switched “off” rather than photobleached. The psFRET technique has some of the FRET imaging advantages 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. Using the same photoswitchable fluorescent proteins, we have also developed a related method called photoswitching anisotropy FRET (psAFRET) for monitoring protein oligomerization via homo-FRET measurements. Although the donors and acceptors are typically fluorescent molecules with different spectra where the donor emission overlaps with the acceptor absorption, homo-FRET can occur between fluorescent molecules of the same type if the emission spectrum overlaps with the absorption spectrum. Since molecules undergoing homo-FRET do not display marked changes in fluorescence spectrum or lifetime, anisotropy measurements are normally employed. Uniquely, our psAFRET method offers the capability to repeatedly monitor the fluorescence anisotropy under both homo-FRET and non-FRET conditions in the same cell. Thus, a critical control measurement is made with every imaging experiment. Data testing the validity of the psFRET and psAFRET approaches to quantify FRET in cells will be presented. In addition, the use of these techniques in imaging protein-protein interactions, protein oligomerization, and fluorescent protein based biosensors will be demonstrated.

 

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