19F labelling of disordered and hybrid proteins for 19F NMR spectroscopy

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216224%3A14740%2F22%3A00125691" target="_blank" >RIV/00216224:14740/22:00125691 - isvavai.cz</a>

Výsledek na webu

<a href="https://www.xray.cz/setkani/abst2022/522.htm" target="_blank" >https://www.xray.cz/setkani/abst2022/522.htm</a>

DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

19F labelling of disordered and hybrid proteins for 19F NMR spectroscopy

Popis výsledku v původním jazyce

<p>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.</p>

Název v anglickém jazyce

19F labelling of disordered and hybrid proteins for 19F NMR spectroscopy

Popis výsledku anglicky

<p>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.</p>

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

2022

Kód důvěrnosti údajů

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