Diffusion-enhanced energy transfer as a live cell probe for cytoplasmic structures




Pinnamaneni, Jaya Pratap

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Diffusion-enhanced resonance energy transfer may be useful as a probe of protein accessibility in bacterial cytoplasmic structures (e.g. microcompartments, nucleoid, inclusion bodies, virus capsids). E. coli takes up luminescent terbium ions under conditions where these ions bind to small, genetically-encoded lanthanide-binding tags (LBT) without toxicity to the cells. Because of their long excited state lifetimes, lanthanides can serve as Förster resonance energy transfer (FRET) donors to energy acceptors that diffuse near them on a millisecond time scale. We designed a test protein called DAL, consisting of an N-terminal dihydrofolate reductase domain, two ankyrin repeats, and a 17-amino acid C-terminal LBT. DAL was expressed in inclusion bodies in E. coli, as indicated by UV luminescence microscopy. Tetramethylrhodamine methyl ester (TMRM), a good FRET acceptor for terbium, was added to the DAL-expressing cells. At a cytoplasmic concentration of 0.5 mM TMRM, there was almost no detectable terbium-to-TMRM FRET. By contrast, purified DAL in free solution had a FRET efficiency of about 0.5 at a TMRM concentration of 0.5 μM. Thus, either the TMRM diffusion coefficient is remarkably low in the cytoplasm (4000-fold lower than in solution), or DAL in inclusion bodies is inaccessible to TMRM. Recent reports indicate that bacterial cytoplasm may form a glass-like state under certain conditions of energy depletion. When we repeated the diffusion-enhanced FRET measurements using log phase cells supplemented with glucose, we observed no change in the apparently low diffusion coefficient. Therefore, we conclude that proteins in inclusion bodies form a diffusion-resistant compartment within the cytoplasm.


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Cytoplasmic, Diffusion-enhanced



Integrative Biology