Unfolding of the recombinant stalk region of GPIbα. (A) Overlaid CD spectra of Ibα-S (solid trace) and Ibα-cSc (dashed) in 50 mM Tris, 50 mM NaCl, and 1 mM DTT; pH 7.4 buffer at 20°C. (B) Chemical denaturation plots of Ibα-S (filled squares) and Ibα-cSc (open squares). (C) Illustration of the optical tweezer setup to measure force-induced unfolding of Ibα-cSc. (D) Force-distance traces showing the force-induced unfolding and refolding of Ibα-cSc from pulling at 100 nm/s. (E) Plot of most probable unfolding force as a function of loading rate. Unfolding forces at 5 different loading rates were plotted as histograms (inset). Each histogram was fitted to a Gaussian curve (inset, solid line) to obtain the most probable force. Uncertainty in force is shown as half of the bin width. The solid line is a linear fit of the data to the Bell-Evans model.46 (F) Unfolding force-extension data (black squares) fitted to the WLC model, yielding a contour length of 22.3 ± 0.2 nm. Extension distances were sorted by unfolding force into 2-pN bins. A histogram of extension of each bin (inset) was fitted to a Gaussian curve (inset, solid line) to find peak extension. Unfolding forces were averaged for each bin (n = 43-149 per bin for unfolding, and 40-64 per bin for refolding). Error bars are 1 SD for force and half-width of the Gaussian fit for extension. Open circles represent refolding force-shortening data, which were treated the same way as the unfolding forces-extension.
