While rare in nature, fluorination of compounds is highly used in industry due to the unique ability of fluorine to change the molecule’s characteristics, such as hydrophobicity, lipophilicity or metabolic stability. Moreover, the addition of fluorine can potentially affect the binding to target proteins, for example by going into multi-orthogonal interactions with fluorophilic environments in the protein, like the protein backbone. As a result, fluorinated compounds are highly used in industry ranging from pharmaceutical over cosmetics to agrochemicals. However, molecular interactions between proteins and these anthropogenic fluorinated molecules remain poorly understood.
Here we aim to close this gap by employing three solute binding proteins (SBP) specific to dicarboxylic acids as a model system to systematically characterize protein-fluorocompound interactions through integrated biophysical and structural approaches. First, utilizing isothermal titration calorimetry we determine the binding of succinate and fumarate as well as their stepwise fluorinated derivatives, revealing that difluoro succinate and monofluoro fumarate do not significantly change the ligand’s binding. However, tetrafluoro succinate leads to a 100-fold decrease in binding. On the contrary, difluoro fumarate results in only one of the SBP to decrease 100-fold in binding and showed no significant effect on the others. To further investigate this effect, we sought to obtain selected 3D protein structure utilizing crystallography. For one SBP we could crystallize it bound to succinate and difluoro succinate, revealing the same binding motif. Based on the crystal structures we performed further analyzations to uncover principles for the effect of additive fluorine to ligand protein interactions.
These findings establish fundamental principles governing protein-fluorocompound recognition and provide a roadmap for engineering fluorinated compound-binding proteins.