Quantifying Stickiness: Thermodynamic Characterization of Intramolecular Domain Interactions To Guide the Design of F枚rster Resonance Energy Transfer Sensors

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• 作者：Laurens H. Lindenburg ; Mantas Malisauskas ; Tari Sips ; Lisanne van Oppen ; Sjors P. W. Wijnands ; Stan F. J. van de Graaf ; Maarten Merkx
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：October 14, 2014
• 年：2014
• 卷：53
• 期：40
• 页码：6370-6381
• 全文大小：437K
• ISSN：1520-4995

文摘

The introduction of weak, hydrophobic interactions between fluorescent protein domains (FPs) can substantially increase the dynamic range (DR) of F枚rster 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)鈭抣inker鈭抏nhanced yellow FP (EYFP) fusion protein, whereas a significantly weaker interaction was observed for the homologous Cerulean/Citrine FRET pair (螖G_o鈥揷p class="stack">0p> = 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鈥搇inker鈥搈Cherry fusion proteins yielded a toolbox of new mOrange鈥搈Cherry combinations that allowed tuning of their intramolecular interaction from very weak (螖G_o鈥揷p class="stack">0p> = 鈭?.39 kCal/mol) to relatively stable (螖G_o鈥揷p class="stack">0p> = 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鈥揅itrine pair by mOrange鈥搈Cherry 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.