Figure 4

Solution structure of proTCC. (a) Elution profile of a solution (100 μl, 1 mg/ml) of proTWT (black line), proTCC (blue line), proTY93A (red line) and proTΔ154-167 (green line) injected onto a Superdex 200 HR 10/30 GL at 0.5 ml/min. Closed (proTWT and proTCC) and open (proTY93A and proTΔ154-167) conformations of prothrombin show a small but significant difference of the retention volume (~0.5 ml), in accordance with their shape in solution. (b) SAXS profile and reconstructed 3D envelope of proTWT (gray) fit with the structure of the closed (protCC, red) or open (proTΔ154-167, blue) conformations of prothrombin. (c) Guided by the structure of protCC, smFRET pairs were designed to cover all possible domain combinations. Probes were attached at residues 34, 101, 120, 160, 210 and 478, which are shown are magenta spheres. Black arrows indicate Cα-Cα distances for 34/478, 34/210 and 101/160, as representative examples. (d) smFRET histograms for proT34/478, proT34/210, proT101/160 and proT101/210. The bottom section of the top graph of each construct depicts the stoichiometry, S, versus FRET efficiency for each diffusing molecule that contains both AF555 and AF647 fluorophores. The upper section shows the one-dimensional efficiency histogram of the molecules in the bottom section. Populations were fit to a single (101/160 and 101/210 FRET couples) or triple (34/478 FRET couple) Gaussian distribution (red lines).