| Popis |
Phosphorylation is the most common post-translational modification that usually alters protein function. 14-3-3 proteins are abundant regulatory proteins that naturally exist in the dimeric oligomeric state. However, stress conditions in the cell trigger phosphorylation of 14-3-3 protein at Ser58, which consequently lead to monomerization of the 14-3-3 protein. Since full and specific phosphorylation at Ser58 for structural-biology studies was not achievable in the past, phosphomimicking mutants were often used. In our project, we optimized preparation of phosphorylated 14-3-3? protein and studied the impact of phosphorylation on its properties in comparison to phosphomimicking and monomeric mutants. We employed a wide range of biophysical techniques and approaches, such as analytical ultracentrifugation, differential scanning calorimetry and fluorimetry, fluorescence assays, isothermal titration calorimetry, mass photometry, nuclear magnetic resonance spectroscopy, mass spectrometry etc., to describe the changes in the oligomeric state, thermal stability, hydrophobicity, structure and interactions with various proteins, peptides as well as small organic compounds. We discovered that in case of 14-3-3 proteins, phosphomimicking mutants insufficiently approximate the effects of phosphorylation, as they differ in the preferred oligomeric state, thermostability and hydrophobicity. Moreover, we revealed that phosphorylation of 14-3-3? elevates intrinsic protein disorder, reduces the affinity towards binding partners, changes the binding stoichiometry and provides additional binding interface. In conclusion, phosphorylation of 14-3-3? protein at Ser58 plays a crucial role in regulation of the protein function. Project acknowledgement: This research was financed by MEYS-CR InterExcellence Inter-Action grant scheme (No. LTAUSA18168), the Czech Science Foundation (No. GF20-05789L), the European Union’s Horizon Europe program (No. 101087124) and the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska Curie grant agreement (No. 873127). We acknowledge CEITEC Proteomics Core Facility, Biomolecular Interactions and Crystallization Core Facility and Josef Dadok National NMR Centre of CIISB, Instruct-CZ Centre, supported by MEYS CR (LM2015043, LM2018127, LM2023042, e-INFRA CZ (ID:90254)), European Regional Development Fund-Project ‘UP CIISB’ (CZ.02.1.01/0.0/0.0/18_046/0015974) and ‘Innovation of Czech Infrastructure for Integrative Structural Biology‘ (No. CZ.02.01.01/00/23_015/0008175). We acknowledge the CEITEC Core Facility Cellular Imaging supported by MEYS CR (LM2018129 Czech-BioImaging).
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