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

Norbert Gašparik*, Aneta Kozeleková, Petr Louša, Jozef Hritz*

Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic

norbert.gasparik@ceitec.muni.cz, jozef.hritz@ceitec.muni.cz

19F NMR has been a very useful complementary approach to traditional techniques - double labelling by 13C and 15N, especially due to the excellent magnetic NMR properties of the 19F isotope. 1) It has a spin ½ 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 [1-3]. Selective 19F isotopic labelling is therefore an outstanding technique for monitoring region-specific changes in protein structure thanks to minimal background signal [4,5].

Here, we present our progress in the preparation of protein samples for 19F NMR measurements, 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 non-optimal 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.

The optimized approaches will be used to study 14-3-3 PPIs and the in vitro formation of tau protein fibrils, a part of Alzheimer’s disease pathology.

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4.       J. G. Bann, J. Pinkner, S. J. Hultgren, C. Frieden. PNAS 99(2), 2002, pp. 709-714.

5.       C. Li, E. A. Lutz, K. M. Slade, R. A. S. Ruf, G-F. Wang, G. J. Pielak. Biochemistry 48, 2009, pp. 8578-8584.

This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic within programme INTER-ACTION (project no. LTAUSA18168). CIISB research infrastructure project LM2018127 funded by MEYS CR is gratefully acknowledged for the financial support of the measurements at the CF Josef Dadok National NMR Centre.