| Autor: |
Bogdanov, Alexey, Gao, Longfei, Dalaloyan, Arina, Zhu, Wenkai, Seal, Manas, Su, Xun-Cheng, Frydman, Veronica, Liu, Yangping, Gronenborn, Angela M., Goldfarb, Daniella |
| Zdroj: |
Physical Chemistry Chemical Physics (PCCP); 11/14/2024, Vol. 26 Issue 42, p26921-26932, 12p |
| Abstrakt: |
19F electron-nuclear double resonance (ENDOR) has emerged as an attractive method for determining distance distributions in biomolecules in the range of 0.7–2 nm, which is not easily accessible by pulsed electron dipolar spectroscopy. The 19F ENDOR approach relies on spin labeling, and in this work, we compare various labels' performance. Four protein variants of GB1 and ubiquitin bearing fluorinated residues were labeled at the same site with nitroxide and trityl radicals and a Gd(III) chelate. Additionally, a double-histidine variant of GB1 was labeled with a Cu(II) nitrilotriacetic acid chelate. ENDOR measurements were carried out at W-band (95 GHz) where 19F signals are well separated from 1H signals. Differences in sensitivity were observed, with Gd(III) chelates providing the highest signal-to-noise ratio. The new trityl label, OXMA, devoid of methyl groups, exhibited a sufficiently long phase memory time to provide an acceptable sensitivity. However, the longer tether of this label effectively reduces the maximum accessible distance between the 19F and the Cα of the spin-labeling site. The nitroxide and Cu(II) labels provide valuable additional geometric insights via orientation selection. Prediction of electron–nuclear distances based on the known structures of the proteins were the closest to the experimental values for Gd(III) labels, and distances obtained for Cu(II) labeled GB1 are in good agreement with previously published NMR results. Overall, our results offer valuable guidance for selecting optimal spin labels for 19F ENDOR distance measurement in proteins. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
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