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- W2050444893 abstract "We previously found that a 17-amino acid lanthanide-binding tag (LBT) expressed as a fusion with a cytoplasmic protein in E. coli takes up Tb3+ in live cells (Biochemistry 50, 6789, 2011). The protein is called DAL after its three domains: dihydrofolate reductase, ankyrin repeats, and LBT. We expressed DAL with a C-terminal 6X-His-tag to create a transition metal binding site for an acceptor of luminescence resonance energy transfer (LRET) from the Tb3+ donor on the LBT. Cu2+ added outside the cells appeared to be transported into the cytoplasm, where it quenched the luminescence of DAL-His-tag similarly to purified DAL-His-tag in free solution. When we expressed DAL lacking a His-tag, we also observed Cu2+ quenching of luminescence, and removal of the highly charged ankyrin domains did not alter Cu2+ quenching. The quenching mechanism is likely to be LRET, because Cu+, which lacks spectral overlap with Tb3+, does not quench DAL luminescence. We conclude that the energy transfer probably occurs by a diffusion-enchanced mechanism. In the small volume of a bacterial cell, freely diffusing donors and acceptors are likely to come in contact during the excited state lifetime of Tb3+ (2 msec). This suggests a possible method for measuring association-dissociation equilibria of large macromolecuar complexes in live bacterial cells. Protein monomers labeled with LRET acceptors (e.g. GFP or FlAsH-tags) can quench DAL donors by diffusional LRET. When associated into a few large assemblies, the acceptors will have much slower diffusion and their locations will no longer be distributed throughout the cytoplasm. We expect that after large assemblies form, diffusion-enhanced LRET will greatly decrease. We plan to test this on E. coli assemblies such as the fiber-forming enzyme CTP synthase, and the ethanolamine utilization microcompartment." @default.
- W2050444893 created "2016-06-24" @default.
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- W2050444893 date "2013-01-01" @default.
- W2050444893 modified "2023-09-26" @default.
- W2050444893 title "Diffusion-Enhanced Luminescence Resonance Energy Transfer in the Cytoplasm of Live Bacterial Cells" @default.
- W2050444893 doi "https://doi.org/10.1016/j.bpj.2012.11.1933" @default.
- W2050444893 hasPublicationYear "2013" @default.
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