Calcium-binding deficiency causes VWF-A2 to be unfolded within HUVECs. (A) Schematic of 4 VWF-A2-FRET proteins, with Venus/YFP and Cerulean/CFP flanking the A2-domain: wild-type VWF-A2, A2-Lock (D1493C, C1669G), R1597W, and D1498M. (B) Wild-type VWF-A2-FRET expressed in HUVECs. Venus signal was measured in first column (excitation = 520 nm; emission = 554-735 nm). Cerulean was excited at 405 nm, and its emission was measured at wavelengths that either do not overlap (em = 437-510 nm, second/Cerulean channel) or that do overlap with Venus (em = 554-735 nm, third/FRET channel). A white rectangular region was marked and fluorescence along its length was measured, in the direction of the orange arrow both for the Cerulean and FRET channels (data in fourth column). Following data capture, Venus was photobleached using a 520-nm laser in the region indicated by the red box (white asterisk). This photobleaching increased Cerulean and depressed FRET signal as shown in the rightmost panel. (C) %FRET was calculated along the length of the white region of interest based on the change in Cerulean signal pre- and postbleaching. This was calculated for all 4 constructs expressed in HUVECs. In each case, arrow indicated the region where photobleaching was performed. A total of 18% to 19% FRET was measured for wild-type and Lock proteins. Calcium-binding mutants (D1498M, R1597W) did not exhibit FRET, suggesting that the proteins are unfolded. (D) HUVECs expressing each of the 4 constructs were analyzed using flow cytometry. FRET ratio (= emitted light in Cerulean/FRET channels) was measured. As seen, the FRET ratio was lower for wild-type VWF-A2 and Lock, compared with the 2 calcium-binding mutants. Thus, R1597W and D1478A remain unfolded in HUVECs.