Methionine-Specific Bioconjugation for Single-Molecule Force Spectroscopy of Cell Surface Proteins

Cell surface proteins play crucial roles in various cellular processes, including intercellular communication, adhesion, and immune responses. However, investigating these proteins using single-molecule force spectroscopy (SMFS) has been hindered by challenges in site-specific protein modification while preserving their native state. Here, we introduce a methionine-specific bioconjugation strategy utilizing a bespoke hypervalent iodine reagent for highly selective, rapid, and robust methionine labeling. Since methionine is often the first amino acid incorporated into proteins via initiator tRNA, this approach enables precise N-terminal labeling and attachment, facilitating more reliable SMFS studies. The resulting covalent linkage remains intact during mechanical unfolding or conformational changes of proteins, with a mechanical stability exceeding 600 pN, allowing accurate measurements before detachment from AFM cantilever tips or cell surfaces. Additionally, this method improves sampling rates and data quality. We successfully applied this technique to light-induced protein printing and natural surface protein studies, demonstrating its potential for advancing protein mechanics research in living cells. This strategy provides significant advantages for SMFS in the study of complex cellular systems.