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

a 2021

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

GAŠPARIK, Norbert, Aneta KOZELEKOVÁ, Petr LOUŠA a Jozef HRITZ

Základní údaje

Originální název

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

Autoři

GAŠPARIK, Norbert (703 Slovensko, domácí), Aneta KOZELEKOVÁ (203 Česká republika, domácí), Petr LOUŠA (703 Slovensko, domácí) a Jozef HRITZ (703 Slovensko, garant, domácí)

Vydání

XXVI. Annual Congress of Czech and Slovak Societies for Biochemistry and Molecular Biology with cooperation of Austrian and German Biochemical Section, 2021

Další údaje

Jazyk

angličtina

Typ výsledku

Konferenční abstrakt

Obor

10608 Biochemistry and molecular biology

Stát vydavatele

Česká republika

Utajení

není předmětem státního či obchodního tajemství

Odkazy

URL

Kód RIV

RIV/00216224:14740/21:00122345

Organizační jednotka

Středoevropský technologický institut

ISBN

978-80-907779-1-0

Klíčová slova česky

14-3-3 protein; 19F NMR

Klíčová slova anglicky

14-3-3 protein; Fluorine NMR; 19F NMR

Změněno: 22. 4. 2022 10:20, Mgr. Norbert Gašparik

Anotace

V originále

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.

Návaznosti

LTAUSA18168, projekt VaV

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

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Selektivní NMR značení jako nástroj pro charakterizaci proteinových komplexů zapojených do neurodegenerativních onemocnění, INTER-ACTION

Citovat

GAŠPARIK, Norbert, Aneta KOZELEKOVÁ, Petr LOUŠA a 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.

@proceedings{1792077,
   author = {Gašparik, Norbert and Kozeleková, Aneta and Louša, Petr and Hritz, Jozef},
   booktitle = {XXVI. Annual Congress of Czech and Slovak Societies for Biochemistry and Molecular Biology with cooperation of Austrian and German Biochemical Section},
   keywords = {14-3-3 protein; Fluorine NMR; 19F NMR},
   language = {eng},
   isbn = {978-80-907779-1-0},
   title = {19F labelling of 14-3-3ζ recombinant protein for 19F NMR spectroscopy},
   url = {http://www.csbmb.cz},
   year = {2021}
}

TY  - CONF
ID  - 1792077
AU  - Gašparik, Norbert - Kozeleková, Aneta - Louša, Petr - Hritz, Jozef
PY  - 2021
TI  - 19F labelling of 14-3-3ζ recombinant protein for 19F NMR spectroscopy
SN  - 9788090777910
KW  - 14-3-3 protein
KW  - Fluorine NMR
KW  - 19F NMR
UR  - http://www.csbmb.cz
N2  - 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.
ER  -

GAŠPARIK, Norbert, Aneta KOZELEKOVÁ, Petr LOUŠA a Jozef HRITZ. 19F labelling of 14-3-3ζ recombinant protein for 19F NMR spectroscopy. In \textit{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.

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