19F labelling of 14-3-3ζ recombinant protein for 19F NMR spectroscopy

• Autoři: GAŠPARIK Norbert; KOZELEKOVÁ Aneta; LOUŠA Petr; HRITZ Jozef
• Rok publikování: 2021
• Druh: Konferenční abstrakty
• Fakulta / Pracoviště MU: Středoevropský technologický institut
• Popis: ¹⁹F NMR has been a very useful complementary approach to traditional techniques - double labelling by ¹³C and ¹⁵N - due to the excellent magnetic properties of the ¹⁹F isotope. 1) It has a spin 1 and strong dipolar coupling (useful in nuclear Overhauser effect spectroscopy), 2) High sensitivity (83% relative to ¹H) and broad chemical shift range (up to 400 ppm), 3) ¹⁹F is 100% abundant in nature and virtually non-present in biologically relevant samples. Selective ¹⁹F isotopic labelling is therefore an outstanding technique to monitor region-specific changes in protein structure owing to minimal background signal. Here, we present our progress in the preparation of 14-3-3 protein samples labelled with ¹⁹F-modified aromatic amino acids (AAs): 5-¹⁹F-Trp, 4-¹⁹F-Phe, and 3-¹⁹F-Tyr. Even though we used identical protocols, different AAs had different incorporation efficiency rates. ¹⁹F tryptophan was readily incorporated with 100% efficiency. However, the extent of incorporation of ¹⁹F phenylalanine and tyrosine ranged only between 30-50%, presumably due to the similar biosynthetic pathways or suboptimal culture conditions. On the other hand, the amount and purity of samples was sufficient for pilot titration experiments, as demonstrated by well-resolved 1D ¹⁹F NMR spectra.

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• Selektivní NMR značení jako nástroj pro charakterizaci proteinových komplexů zapojených do neurodegenerativních onemocnění