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@proceedings{1848277,
author = {Gašparik, Norbert and Kozeleková, Aneta and Crha, Radek and Hritz, Jozef},
booktitle = {XVIII Discussions in Structural Molecular Biology and 5th User Meeting of CIISB},
keywords = {14-3-3 protein; Fluorine NMR; 19F NMR; Tau protein},
language = {eng},
title = {19F labelling of disordered and hybrid proteins for 19F NMR spectroscopy},
url = {https://www.xray.cz/setkani/abst2022/522.htm},
year = {2022}
}
TY - CONF ID - 1848277 AU - Gašparik, Norbert - Kozeleková, Aneta - Crha, Radek - Hritz, Jozef PY - 2022 TI - 19F labelling of disordered and hybrid proteins for 19F NMR spectroscopy KW - 14-3-3 protein KW - Fluorine NMR KW - 19F NMR KW - Tau protein UR - https://www.xray.cz/setkani/abst2022/522.htm N2 - 19F NMR has been a 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, 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 hybrid and disordered protein samples, labelled with 19F modified aromatic amino acids (AAs), for use in 19F NMR spectroscopy measurements. When using identical protocols, different AAs proved to exhibit different incorporation efficiency rates. 19F tryptophan was readily incorporated with 100% efficiency. The extent of incorporation of 19F phenylalanine and tyrosine first ranged only between 30-50%, presumably due to similar biosynthetic pathways. Extensive optimisation of culture media, amount of labelled amino acid, wash and recovery period, and bacterial strains were utilised to increase the labelling rate of 14-3-3 and Tau proteins by the mentioned amino acids. In our efforts, we enhanced the labelling efficiency rate around twofold, as confirmed by MS/MS spectrometry and 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. ER - GAŠPARIK, Norbert, Aneta KOZELEKOVÁ, Radek CRHA a Jozef HRITZ. 19F labelling of disordered and hybrid proteins for 19F NMR spectroscopy. In \textit{XVIII Discussions in Structural Molecular Biology and 5th User Meeting of CIISB}. 2022.
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