Site‐Specific Incorporation of Fluorinated Prolines into Proteins and Their Impact on Neighbouring Residues

The incorporation of fluorinated amino acids into proteins provides new opportunities to study biomolecular structure-function relationships in an elegant manner. The available strategies to incorporate the majority of fluorinated amino acids are not site-specific or imply important structural modifications. Here, we present a chemical biology approach for the site-specific incorporation of three commercially available C^γ-modified fluoroprolines that has been validated using a non-pathogenic version of huntingtin exon-1 (HttExon-1). ¹⁹F, ¹H and ¹⁵N NMR chemical shifts measured for multiple variants of HttExon-1 indicated that the trans/cis ratio was strongly dependent on the fluoroproline variant and the sequence context. By isotopically labelling the rest of the protein, we have shown that the extent of spectroscopic perturbations to the neighbouring residues depends on the number of fluorine atoms and the stereochemistry at C^γ, as well as the isomeric form of the fluoroproline. We have rationalized these observations by means of extensive molecular dynamics simulations, indicating that the observed atomic chemical shift perturbations correlate with the distance to fluorine atoms and that the effect remains very local. These results validate the site-specific incorporation of fluoroprolines as an excellent strategy to monitor intra- and intermolecular interactions in disordered proline-rich proteins.