MT force spectroscopy reveals conformational change in the D'D3 domain. (A) Extension-time trace of a VWF dimer exhibiting fast, reversible transitions between 3 distinct states. After checking for A2 domain unfolding at 11 pN (blue inset, unfolding events indicated by arrows), the force is decreased from 12 to 6 pN to systematically study the transitions between 3 states, separated by ≈7.5 nm. The population in the respective states shifts with decreasing force, with an increasing fraction of time spent in the lower extension levels at lower forces. At forces around 8 pN, transitions from the M state to the T or B state are equally likely (orange inset). In the inset, the 3 states (top, middle, and bottom) are indicated by dashed lines. The histogram is fit with a triple Gaussian function (black line; Table 1, Equation 10). Raw data recorded at 58 Hz (gray line, inset) and smoothed data at 5 Hz (main panel and orange line in the inset). (B) Histogram of contour length transformed increment of the wild-type dimer and a modified dimer with an additional 20-aa-long linker insertion into the naturally occurring flexible sequence between VWD3 and C8-3 (position of the linker insert indicated in Civ). Lines are Gaussian fits (Table 1, Equation 8); the mean ± standard deviation are L_C,wt = 19.0 ± 2.4 nm and L_C,linker = 32.4 ± 4.1 nm. (C) Force-probe molecular dynamics simulations reveal the molecular mechanism of the transitions in the D’D3 domain. (i) Crystal structure of the D'D3 domain with TIL' (gray), E' (purple), VWD3 (blue), C8-3 (red), TIL3 (green), and E3 (orange).¹⁶  Termini are marked with cyan spheres. External forces F are applied on them in opposite directions with a pulling velocity of 0.025 m/s. (ii-iv) Under the influence of force, E3, TIL3, and C8-3 are “peeled” off the large VWD3 submodule. Red arrow indicates position of the linker insert. The panels at the left side indicate the normalized average number of contacts between VWD3 and the indicated subdomain as a function of the inter-termini extension. A decrease of contacts indicates the opening of the interface between VWD3 and the respective domains. Data shown are with neutral histidine residues (HIS(0)) or with histidines protonated (HIS(+)). The contour length increase estimates from the simulations on the basis of the force-distance profiles (supplemental Figure 3) are 16.4 ± 0.3 nm (HIS(0): vertical gray line) and 17.1 ± 0.3 nm (HIS(+): vertical black line).