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- W2144951150 abstract "The introduction of weak, hydrophobic interactions between fluorescent protein domains (FPs) can substantially increase the dynamic range (DR) of Förster resonance energy transfer (FRET)-based sensor systems. Here we report a comprehensive thermodynamic characterization of the stability of a range of self-associating FRET pairs. A new method is introduced that allows direct quantification of the stability of weak FP interactions by monitoring intramolecular complex formation as a function of urea concentration. The commonly used S208F mutation stabilized intramolecular FP complex formation by 2.0 kCal/mol when studied in an enhanced cyan FP (ECFP)−linker−enhanced yellow FP (EYFP) fusion protein, whereas a significantly weaker interaction was observed for the homologous Cerulean/Citrine FRET pair (ΔGo–c0 = 0.62 kCal/mol). The latter effect could be attributed to two mutations in Cerulean (Y145A and H148D) that destabilize complex formation with Citrine. Systematic analysis of the contribution of residues 125 and 127 at the dimerization interface in mOrange–linker–mCherry fusion proteins yielded a toolbox of new mOrange–mCherry combinations that allowed tuning of their intramolecular interaction from very weak (ΔGo–c0 = −0.39 kCal/mol) to relatively stable (ΔGo–c0 = 2.2 kCal/mol). The effects of these mutations were also studied by monitoring homodimerization of mCherry variants using fluorescence anisotropy. These mutations affected intramolecular and intermolecular domain interactions similarly, although FP interactions were found to be stronger in the latter. The knowledge thus obtained allowed successful construction of a red-shifted variant of the bile acid FRET sensor BAS-1 by replacement of the self-associating Cerulean–Citrine pair by mOrange–mCherry variants with a similar intramolecular affinity. Our findings thus allow a better understanding of the subtle but important role of intramolecular domain interactions in current FRET sensors and help guide the construction of new sensors using modular design strategies." @default.
- W2144951150 created "2016-06-24" @default.
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- W2144951150 date "2014-09-26" @default.
- W2144951150 modified "2023-09-26" @default.
- W2144951150 title "Quantifying Stickiness: Thermodynamic Characterization of Intramolecular Domain Interactions To Guide the Design of Förster Resonance Energy Transfer Sensors" @default.
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- W2144951150 doi "https://doi.org/10.1021/bi500433j" @default.
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