Single-molecule studies on VWF A1 and A2 domains. (A-D) Adapted from Zhang et al⁴³  with permission. (A) Schematic diagram of how the A2 domain (colored in rainbow as in Figure 5A) is held between 800-bp double-stranded DNA handles and tethered to 2 beads in a laser trap (left) and micropipette (right). DNA is covalently linked through disulfide bridges to Cys residues mutationally added at the N and C termini of A2. DNA handles have biotin and digoxigenin (Dig) tags at opposite ends for binding to beads functionalized with streptavidin and digoxigenin antibody. Force is applied by micropipette movement (right), and measured by bead displacement in the laser trap (left). The sine qua non of single molecule data are measurement of single molecule events; other types of events are recorded and they must be discarded using fiduciary markers. DNA handles provide a single molecule signature, that is, a plateau at 67 pN at a transition from B to S DNA. Furthermore, adsorption to beads, cantilever tips, and substrates is prevented by holding proteins away from them with DNA handles. (B) Three representative cycles of force increase, decrease, and clamping at a constant low level to enable A2 refolding. (C) Traces of force vs tether extension in the force increase phases of cycles ii and iii in panel B. An abrupt unfolding event is seen in ii and not iii. It is inferred that A2 was unfolded at the beginning of iii. (D) Two representative traces showing ADAMTS13 cleavage in presence of enzyme (1) and no cleavage in absence of enzyme (2). In each trace unfolding of A2 is seen, and A2 is returned to a clamped force of 5 pN. Cleavage of the tether returns force to 0. (E-G). Repeated measurement of GPIbα and VWF A1 domain binding and unbinding in a single molecule ReaLiSM construct. Modified from Kim et al⁷¹  with permission. (E) Schematic. The ReaLiSM contains from N to C the A1 domain, a 43-residue polypeptide linker, and GPIbα, and is expressed as a secreted protein in mammalian cells. Cysteines are included at the N and C termini for disulfide linkage to DNA handles, which are coupled to beads as in panel A. (F) One representative cycle. Unbinding and rebinding are measured as abrupt changes in tether extension during pulling (red) and relaxation (black). (G) Schematic model of A1- GPIbα flex-bond. The model reflects 2 different pathways for receptor-ligand dissociation⁷¹  and association (J. S. Kim, N. E. Hudson, and T.A.S., unpublished observations), with a slower dissociating and faster associating state induced by force. A1 (cyan) and GPIbα (magenta) are subjected to tensile force at the N and C termini of the ReaLiSM construct (arrows), and after dissociation, also at the junctions with the polypeptide linker.