Fig. 1.

Attachment protocol for highly parallel force spectroscopy on proteins in MT. (A) Schematic of the strategy for tethering a protein of interest between the bottom glass slide of the flow cell and a magnetic bead (not to scale). An ELP linker with a single cysteine at its N terminus is coupled to the amino-silanized glass slide via a small-molecule NHS–maleimide cross-linker. After covalent coupling of CoA–biotin to the ybbr-tag at the C terminus of the protein in a bulk reaction catalyzed by sfp phosphopantetheinyl transferase, the protein is covalently ligated to the ELP linker via one (or more) glycines at its N terminus in a reaction mediated by sortase A, which selectively recognizes the C-terminal LPETGG motif of the ELP linker. Finally, a streptavidin-coated magnetic bead is bound to the biotinylated protein via the high-affinity biotin–streptavidin interaction. Red and gray double arrows indicate covalent and noncovalent bonds, respectively. Forces are exerted on the magnetic bead by permanent magnets positioned above the flow cell. Nonmagnetic polystyrene beads baked onto the surface are used as reference beads for drift correction. (B) Representative field of view. Yellow boxes indicate ∼60 beads marked for tracking. (B, Inset) The enlarged image of 1 bead shows the diffraction ring pattern used for 3D bead tracking. (C) Example tether extension time traces showing the characteristic 3-state unfolding pattern of ddFLN4. All traces shown were recorded in parallel from different beads within the same field of view at a constant force of 21 pN.