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