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. 2016 Jul 26;111(2):312–322. doi: 10.1016/j.bpj.2016.06.022

Figure 3.

Figure 3

Conformation of dimeric VWF constructs under varied pH and ionic conditions. (A) Representative AFM image of full-length VWF dimers (D′D3-CK) adsorbed onto a functionalized mica surface from buffer solution without divalent ions and adjusted to pH 6.2. Arrows mark the positions of CK domains. A large fraction (>50%) of dimers exhibited a compact conformation with fully formed C-terminal stems. Insets show examples of a flexible dimer with a partially formed stem and a compact dimer with a fully formed stem, respectively. Numbers indicate values of the normalized stem length. Scale bar is 100 nm; range of color scale is 2.4 nm. (B) Representative AFM image of VWF dimers with a deletion of the D4 domain (delD4 dimers), adsorbed onto a functionalized mica substrate from buffer solution adjusted to pH 6.8 and containing divalent ions. White arrows mark the positions of CK domains. Throughout the whole probed pH range, nearly all delD4 dimers exhibited a flexible conformation. The green arrow indicates an occasional compact dimer. Scale bar is 100 nm; range of color scale is 2.4 nm. (C) Ratio of compact VWF dimers as a function of the pH. Only dimers with a normalized stem length above 1 were assigned as compact. Shown are data for full-length dimers in the presence (blue circles) and absence (red triangles) of divalent ions, and for delD4 dimers in the presence of divalent ions (brown squares). The dashed red line is a sigmoidal fit (half-maximum value at pH 6.6) to the data obtained for full-length dimers in the absence of divalent ions, i.e., under conditions that obstruct the formation of the strong intermonomer interaction mediated by the D4 domain (13). Error bars represent Poisson noise (1 SD). For each data point, n ≥ 100 dimers were analyzed. (D) SAXS profiles in Kratky representation (top) and computed distance distribution functions P(r) (bottom) for dimeric A1-CK constructs in the presence of divalent ions at pH 6.2 (dark blue) and pH 7.4 (light blue), and in the presence of 10 mM EDTA at pH 7.4 (red). To see this figure in color, go online.