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Studying Proteins in Live
Cells using Single Molecule Fluorescence Resonance Energy Transfer The goal of this project is to employ single molecule fluorescence resonance energy transfer (smFRET) combined with total internal reflection microscopy to study protein dynamics in live cells. We aim to achieve this by strategically dye-labeling recombinantly expressed SNARE proteins (specifically SNAP-25 and SNAP-29) and microinjecting them into cultured cells grown on coverslips. Our hope is that in vivo smFRET will allow us to know the conformational state of proteins while simultaneously allowing us to study their location and dynamic properties.
Example of smFRET
signal from dye-labeled SNAP-25 in a live cell. Red is the acceptor dye and green the donor. The single step photobleach
of the red and anticorrelation of the green is
indicative of smFRET.
SNAP-25 is part of the heterotrimeric SNARE complex critical in vesicle fusion. Dyes are placed such that the protein will give high FRET in complex (center) and low or zero FRET out of complex (right). |
