Pulling the engaged A1 domain of VWF induces unfolding of a domain in the full-length GPIb-IX complex. (A) A diagram of GPIb-IX illustrating the experimental setup for the optical tweezer single-molecule force measurement. The BioTag sequence that is specifically recognized and biotinylated by Escherichia coli biotin ligase was either appended to the C-terminus of GPIX cytoplasmic domain (as shown) or placed into the juxtamembrane region of the GPIbα cytoplasmic domain. The biotinylated GPIb-IX was expressed in transfected cells, solubilized in the Triton X-100–containing lysis buffer, and eventually immobilized on the streptavidin-coated bead. Recombinant VWF-A1 was linked through a DNA handle to a polystyrene bead that was placed in an optical trap as described before.³  Individual domains of GPIbα are marked on the left. (B) The cytoplasmic sequence of GPIX-biotag showing the appended site of biotinylation. A c-myc immunotag and a BioTag sequence (underlined) are attached to the C-terminal end of GPIX. Residues in the GPIX transmembrane domain are marked by a gray box. (C) A single-molecule force-distance trace illustrating the unfolding of MSD before the detachment of VWF-A1 from GPIb-IX. The inset highlights the observed unfolding event.