Influence of Fluorination on Single-Molecule Unfolding and Rupture Pathways of a Mechanostable Protein Adhesion Complex

Abstract

AbstractFluorination of proteins by cotranslational incorporation of non-canonical amino acids is a valuable tool for enhancing biophysical stability. Despite many prior studies investigating the effects of fluorination on equilibrium stability, its influence on non-equilibrium mechanical stability remains unknown. Here, we used single-molecule force spectroscopy (SMFS) with the atomic force microscope (AFM) to investigate the influence of fluorination on unfolding and unbinding pathways of a mechanically ultrastable bacterial adhesion complex. We assembled modular polyproteins comprising the tandem dyad XModule-Dockerin (XMod-Doc) bound to a globular Cohesin (Coh) domain. By applying tension across the binding interface, and quantifying single-molecule unfolding and rupture events, we mapped the energy landscapes governing the unfolding and unbinding reactions. We then used sense codon suppression to substitute trifluoroleucine (TFL) in place of canonical leucine (LEU) globally in XMod-Doc, or selectively within the Doc subdomain of a mutant XMod-Doc. Although TFL substitution thermally destabilized XMod-Doc, it had little effect on XMod-Doc:Coh binding affinity at equilibrium. When we mechanically dissociated global TFL-substituted XMod-Doc from Coh, we observed the emergence of a new unbinding pathway with a lower energy barrier. Counterintuitively, when fluorination was restricted to Doc, we observed mechano-stabilization of the non-fluorinated neighboring XMod domain. These results suggest that intramolecular deformation networks can be modulated by fluorination, and further highlight significant differences between equilibrium thermostability, where all constructs were destabilized, and non-equilibrium mechanostability, where XMod was strengthened. Future work is poised to investigate the influence of non-natural amino acids on mechanically-accelerated protein unfolding and unbinding reaction pathways.