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

Abstract

AbstractSingle‐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.