Fig. 6: The second HS-binding site in Shh facilitates cross-linking and switching between heparins.

a–c QCM-D binding assays analogous to Fig. 5, for Shh (a), Shh^K/A (b), and Shh^K/E (c), with an added step of competition with soluble heparin. d Overlays of frequency responses as shown in (a–c) demonstrate a reduced binding rate and increased unbinding rate in the wash buffer of Shh^K/E, and reduced unbinding of Shh^K/A and Shh^K/E upon competition with soluble heparin, compared to Shh. e Overlay of associated dissipation shifts as shown in (a–c) evidences reduced rigidification of the heparin layer by Shh^K/A, and even more so by Shh^K/E, compared to Shh, demonstrating that the second HS-binding site is essential for HS cross-linking. f Overlay of frequency responses (relative to full protein elution) from the start of soluble heparin-mediated protein elution (t = 0) until t = 40 min after elution start evidences an overall reduced elution rate for Shh^K/A and Shh^K/E compared to Shh, thus demonstrating that the second HS-binding site of Shh promotes rapid HS switching.