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

GAŠPARIK, Norbert, Aneta KOZELEKOVÁ, Petr LOUŠA and Jozef HRITZ. 19F labelling of 14-3-3ζ recombinant protein for 19F NMR spectroscopy. In XXVI. Annual Congress of Czech and Slovak Societies for Biochemistry and Molecular Biology with cooperation of Austrian and German Biochemical Section. 2021. ISBN 978-80-907779-1-0.

Basic information

• Original name: 19F labelling of 14-3-3ζ recombinant protein for 19F NMR spectroscopy
• Authors: GAŠPARIK, Norbert (703 Slovakia, belonging to the institution), Aneta KOZELEKOVÁ (203 Czech Republic, belonging to the institution), Petr LOUŠA (703 Slovakia, belonging to the institution) and Jozef HRITZ (703 Slovakia, guarantor, belonging to the institution).
• Edition: XXVI. Annual Congress of Czech and Slovak Societies for Biochemistry and Molecular Biology with cooperation of Austrian and German Biochemical Section, 2021.

Other information

• Original language: English
• Type of outcome: Conference abstract
• Field of Study: 10608 Biochemistry and molecular biology
• Country of publisher: Czech Republic
• Confidentiality degree: is not subject to a state or trade secret
• WWW: URL
• RIV identification code: RIV/00216224:14740/21:00122345
• Organization unit: Central European Institute of Technology
• ISBN: 978-80-907779-1-0
• Keywords (in Czech): 14-3-3 protein; 19F NMR
• Keywords in English: 14-3-3 protein; Fluorine NMR; 19F NMR

Abstract

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 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. 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.

Links

• LTAUSA18168, research and development project: Name: Selektivní NMR značení jako nástroj pro charakterizaci proteinových komplexů zapojených do neurodegenerativních onemocnění

Investor: Ministry of Education, Youth and Sports of the CR, Selective NMR labelling as a tool for characterization of protein complexes involved in neurodegenerative diseases., INTER-ACTION