Direct Comparison of Lysine versus Site-Specific Protein Surface Immobilization in Single-Molecule Mechanical Assays**

Single-molecule force spectroscopy (SMFS) is powerful for studying folding states and mechanical properties of proteins, however, it requires protein immobilization onto force-transducing probes such as cantilevers or microbeads. A common immobilization method relies on coupling lysine residues to carboxylated surfaces using 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS). Because proteins typically contain many lysine groups, this strategy results in a heterogeneous distribution of tether positions. Genetically encoded peptide tags (e.g., ybbR) provide alternative chemistries for achieving site-specific immobilization, but thus far a direct comparison of site-specific vs. lysine-based immobilization strategies to assess effects on the observed mechanical properties was lacking. Here, we compared lysine- vs. ybbR-based protein immobilization in SMFS assays using several model polyprotein systems. Our results show that lysine-based immobilization results in significant signal deterioration for monomeric streptavidin-biotin interactions, and loss of the ability to correctly classify unfolding pathways in a multipathway Cohesin-Dockerin system. We developed a mixed immobilization approach where a site-specifically tethered ligand was used to probe surface-bound proteins immobilized through lysine groups, and found partial recovery of specific signals. The mixed immobilization approach represents a viable alternative for mechanical assays on in vivo-derived samples or other proteins of interest where genetically encoded tags are not feasible.