title: Quantifying stickiness: thermodynamic characterization of intramolecular domain interactions to guide the design of förster resonance energy transfer sensors.
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 (ΔG0(o-c) = 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 (ΔG0(o-c) = .0.39 kCal/mol) to relatively stable (ΔG0(o-c) = 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.
authors.affiliation: Laboratory of Chemical Biology and Institute of Complex Molecular Systems (ICMS), Department of Biomedical Engineering, Eindhoven University of Technology , Eindhoven, The Netherlands.
authors.forename: Laurens H
authors.initials: LH
authors.lastname: Lindenburg
authors.forename: Mantas
authors.initials: M
authors.lastname: Malisauskas
authors.forename: Tari
authors.initials: T
authors.lastname: Sips
authors.forename: Lisanne
authors.initials: L
authors.lastname: van Oppen
authors.forename: Sjors P W
authors.initials: SP
authors.lastname: Wijnands
authors.forename: Stan F J
authors.initials: SF
authors.lastname: van de Graaf
authors.forename: Maarten
authors.initials: M
authors.lastname: Merkx
mesh_terms: D019943:Amino Acid Substitution; D001647:Bile Acids and Salts / Q000737:chemistry; D015374:Biosensing Techniques; D005454:Fluorescence Polarization; D031541:Fluorescence Resonance Energy Transfer; D057927:Hydrophobic and Hydrophilic Interactions; D008164:Luminescent Proteins / Q000737:chemistry; D011485:Protein Binding; D011489:Protein Denaturation; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D055550:Protein Stability; D013816:Thermodynamics; D014508:Urea / Q000737:chemistry; D000097573:Red Fluorescent Protein
chemical_list: D001647:Bile Acids and Salts; D008164:Luminescent Proteins; D014508:Urea; D000097573:Red Fluorescent Protein