Electrostatic Contribution to ¹⁹F Chemical Shifts in Fluorotryptophans in Proteins

DFT calculations indicate that the ¹⁹F chemical shifts of aromatic rings containing single fluorine substituents are sensitive to the electric fields and electric field gradients at the position of the fluorine atom. The present work explores whether long-range structure restraints can be gained from changes in ¹⁹F chemical shifts following mutations of charged to uncharged residues. ¹⁹F chemical shifts of fluorotryptophan residues were measured in two different proteins, GB1 and the NT* domain, following mutations of single asparagine residues to aspartic acid. Four different versions of fluorotryptophan were investigated, including 4-, 5-, 6-, and 7-fluorotryptophan, which were simultaneously installed by cell-free protein synthesis using 4-, 5-, 6-, and 7-fluoroindole as precursors for the tryptophan synthase present in the S30 extract. For comparison, the ¹H chemical shifts of the corresponding nonfluorinated protein mutants produced with ¹³C-labeled tryptophan were also measured. The results show that the ¹⁹F chemical shifts respond more sensitively to the charge mutations than the ¹H chemical shifts in the nonfluorinated references, but the chemical shift changes were much smaller than predicted by DFT calculations of fluoroindoles in the electric field of a partial charge in vacuum, indicating comprehensive dielectric shielding by water and protein. No straightforward correlation with the location of the charge mutation could be established.