Figure 1.

Handle attachment procedure for single molecule manipulations. The DNA handles are first linked to the MBP‐SpyCatcher fusion using the traditional thiol chemistry approach. Separately the target protein (in this case SpyTag‐GlpG) is prepared with added SpyTag peptides (genetically encoded). Upon simple mixing, SpyTag‐GlpG becomes covalently linked to the MBP‐SpyCatcher‐DNA via an isopeptide bond (see box inset). For unfolding experiments with GlpG, we employ bicelles to provide bilayer conditions, which are fully compatible with the attachment method. The DNA‐conjugated GlpG can then be tethered between the glass support and magnetic bead via the biotin/neutravidin and dig/antidig linkages, and subject to mechanical force.