| Autor: |
Orton, Henry W., Qianzhu, Haocheng, Abdelkader, Elwy H., Habel, Edan I., Tan, Yi Jiun, Frkic, Rebecca L., Jackson, Colin J., Huber, Thomas, Otting, Gottfried |
| Zdroj: |
Journal of the American Chemical Society; 20210101, Issue: Preprints |
| Abstrakt: |
Fluorine atoms are known to display scalar 19F–19F couplings in nuclear magnetic resonance (NMR) spectra when they are sufficiently close in space for nonbonding orbitals to overlap. We show that fluorinated noncanonical amino acids positioned in the hydrophobic core or on the surface of a protein can be linked by scalar through-space 19F–19F (TSJFF) couplings even if the 19F spins are in the time average separated by more than the van der Waals distance. Using two different aromatic amino acids featuring CF3groups, O-trifluoromethyl-tyrosine and 4-trifluoromethyl-phenylalanine, we show that 19F–19F TOCSY experiments are sufficiently sensitive to detect TSJFFcouplings between 2.5 and 5 Hz in the 19 kDa protein PpiB measured on a two-channel 400 MHz NMR spectrometer with a regular room temperature probe. A quantitative Jevolution experiment enables the measurement of TSJFFcoupling constants that are up to five times smaller than the 19F NMR line width. In addition, a new aminoacyl-tRNA synthetase was identified for genetic encoding of N6-(trifluoroacetyl)-l-lysine (TFA-Lys) and 19F–19F TOCSY peaks were observed between two TFA-Lys residues incorporated into the proteins AncCDT-1 and mRFP despite high solvent exposure and flexibility of the TFA-Lys side chains. With the ready availability of systems for site-specific incorporation of fluorinated amino acids into proteins by genetic encoding, 19F–19F interactions offer a straightforward way to probe the spatial proximity of selected sites without any assignments of 1H NMR resonances. |
| Databáze: |
Supplemental Index |
| Externí odkaz: |
|
