Fluorine atoms are known to display scalar 19Fâ19F couplings in nuclear magnetic resonance (NMR) spectra when they are sufficiently close in space for nonbonding orbitals to overlap. This phenomenon can be exploited to measure intra- and inter-residue distances in proteins, providing a powerful tool for investigating protein structure and function. Despite this, little effort has been invested in characterising the effects of fluorine substitutions in a protein. We have utilised E. coli peptidyl-prolyl cis/trans-isomerase B (PpiB) as a model protein for incorporation of fluorinated noncanonical amino acids to probe the detection of specific contacts. Six different fluorinated amino acids, including Tyrosine, Phenylalanine, Leucine, and Valine, were incorporated into PpiB. Crystal structures ranging from 2.0â1.22 Ă resolution were solved for each of these, which provide insight into the limited structural perturbations elicited by the fluorine substitutions. The CF3 groups on the tyrosine and phenylalanine residues provide a unique probe for detecting specific interactions within the protein, while the fluorinated leucines and valines provide a non-invasive way to monitor protein dynamics. Our results demonstrate the potential of using fluorinated noncanonical amino acids as powerful tools for investigating protein structure and function.