The main role of dimeric 14-3-3 proteins is to modulate the activity of several hundred binding partners by interacting with phosphorylated residues of the partner proteins, often located in disordered regions. The inherent flexibility or large size of 14-3-3 complexes hampers their structural characterization by X-ray crystallography, cryo-electron microscopy (EM) and traditional solution nuclear magnetic resonance (NMR) spectroscopy. Here, we employ solution 1D 19F-Trp NMR spectroscopy to characterize substrate binding and dimerization of 14-3-3 proteins, focusing on 14-3-3ζ - an abundant human isoform as an example. Both conserved Trp residues are located in distinct functionally important sites - the dimeric interface and the ligand-binding groove. We substituted them by 5F-Trp, thereby introducing a convenient NMR probe. Fluorination of the two Trp did not impact the stability and interaction properties of 14-3-3ζ in a substantive manner, permitting to carry out 19F NMR experiments to assess 14-3-3's structure and behavior. Importantly, 5F-Trp228 reports on binding of substrates in the amphipathic binding groove of 14-3-3ζ and permitted to distinguish distinct recognition modes. Thus, we established that 19F NMR is a powerful approach to evaluate the binding of partner proteins to 14-3-3 and to characterize the properties of the resulting complexes.

Central European Institute of Technology Masaryk University Kamenice 5 Brno 625 00 Czechia; National Centre for Biomolecular Research Faculty of Science Masaryk University Kamenice 5 Brno 625 00 Czechia

Central European Institute of Technology Masaryk University Kamenice 5 Brno 625 00 Czechia; National Centre for Biomolecular Research Faculty of Science Masaryk University Kamenice 5 Brno 625 00 Czechia; Department of Chemistry Faculty of Science Masaryk University Kamenice 5 Brno 625 00 Czechia

Department of Structural Biology University of Pittsburgh School of Medicine 1051 Biomedical Science Tower 3 3501 5th Ave Pittsburgh PA 15261 USA

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