Publication details

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

Authors

GAŠPARIK Norbert KOZELEKOVÁ Aneta LOUŠA Petr HRITZ Jozef

Year of publication 2021
Type Conference abstract
MU Faculty or unit

Central European Institute of Technology

Citation
Description 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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