Abstract
αβ T cells are mechanosensors that leverage bioforces during immune surveillance for highly sensitive and specific antigen discrimination. Single-molecule studies are used to profile the initial TCRαβ-pMHC binding event, and various biophysical parameters can be identified. Isolating purified TCRαβ and pMHC molecules on a coverslip allows for direct measurements of the kinetics and conformational changes in the system and removes cellular components along the load pathway that may interfere with or mask subtle changes. Optical tweezers provide high resolution position and force information that map the bonding profile, including catch bond, and the ability to measure distinct conformational changes driven by forces. The present method describes the single-molecule optical tweezers assay setup, considerations, and execution. This model can be used for various TCR-pMHC pairs or expanded to measure a wide variety of receptor-ligand interactions operative in multiple biological systems.