Time-Resolved Luminescence Resonance Energy Transfer Imaging of Protein Interactions in Living Cells.
Rajapakse, Harsha E.
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Time-resolved, Luminescence Resonance Energy Transfer (LRET) imaging using lanthanide probes was shown to be a faster and more sensitive means to visualize dynamic interactions of proteins in living cells when compared to conventional steady state imaging of Förster Resonance Energy Transfer (FRET). This dissertation provides a theoretical description of LRET imaging with lanthanide probes as well as experimental results showing: i) syntheses of luminescent terbium complexes that bind specifically and stably to Escherichia coli dihydrofolate reductase (eDHFR) fusion proteins; ii) that terbium complexes can be delivered into the cytoplasm of living cells; and iii) that intracellular interactions between transgenically expressed eDHFR fusion proteins (labeled with terbium complexes) and transgenically expressed GFP fusion proteins can be imaged by detecting terbium-to-GFP LRET with a time-resolved fluorescence microscope. This dissertation also describes detailed protocols for practical implementation of the experimental methods. Time-resolved LRET microscopy was used to detect interactions between two epithelial tight junction protein domains: the first PDZ domain of ZO-1, fused to eDHFR and the cytoplasmic, C-terminal YV domain of claudin-1, fused to GFP. Interactions were detected in single microscope images at sub-second time scales, and a highly significant (P < 10-6), six-fold difference between the mean, donor-normalized LRET signal from cells expressing interacting fusion proteins and from control cells expressing non-interacting mutants was observed. The results show that time-resolved, LRET microscopy with a selectively targeted, luminescent terbium protein label affords improved speed and sensitivity over conventional FRET methods for a variety of live-cell imaging and screening applications.