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

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216224%3A14740%2F21%3A00122345" target="_blank" >RIV/00216224:14740/21:00122345 - isvavai.cz</a>

Výsledek na webu

<a href="http://www.csbmb.cz" target="_blank" >http://www.csbmb.cz</a>

DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

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

Popis výsledku v původním jazyce

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.

Název v anglickém jazyce

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

Popis výsledku anglicky

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.

Druh

O - Ostatní výsledky

CEP obor

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OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

<a href="/cs/project/LTAUSA18168" target="_blank" >LTAUSA18168: Selektivní NMR značení jako nástroj pro charakterizaci proteinových komplexů zapojených do neurodegenerativních onemocnění</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2021

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů