19F labelling of disordered and hybrid proteins for 19F NMR spectroscopy
The result's identifiers
Result code in 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>
Result on the web
<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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Alternative languages
Result language
angličtina
Original language name
19F labelling of disordered and hybrid proteins for 19F NMR spectroscopy
Original language description
<p><sup>19</sup>F NMR has been a useful complementary approach to traditional techniques - double labelling by <sup>13</sup>C and <sup>15</sup>N, especially due to the excellent magnetic NMR properties of the <sup>19</sup>F isotope. 1) It has a spin ½ and strong dipolar coupling, 2) High sensitivity (83% relative to <sup>1</sup>H) and broad chemical shift range (up to 400 ppm), 3) <sup>19</sup>F is 100% abundant in nature and virtually non-present in biologically relevant samples [1-3]. Selective <sup>19</sup>F 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 <sup>19</sup>F modified aromatic amino acids (AAs), for use in <sup>19</sup>F NMR spectroscopy measurements. When using identical protocols, different AAs proved to exhibit different incorporation efficiency rates. <sup>19</sup>F tryptophan was readily incorporated with 100% efficiency. The extent of incorporation of <sup>19</sup>F 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 <sup>19</sup>F 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>
Czech name
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Czech description
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Classification
Type
O - Miscellaneous
CEP classification
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OECD FORD branch
10608 - Biochemistry and molecular biology
Result continuities
Project
<a href="/en/project/LTAUSA18168" target="_blank" >LTAUSA18168: Selective NMR labelling as a tool for characterization of protein complexes involved in neurodegenerative diseases.</a><br>
Continuities
P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)
Others
Publication year
2022
Confidentiality
S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů