Figure S4. Structures of Helix 1 and the Hex fragment and control specimens for light-scattering-based flux assay.

(A) Secondary structure prediction of the second half of the fragment F1 contains the Helix 1 (CHGB82-106). (B) Sequence alignment of the C-terminal half of the F1 fragments from different species (mCHGB: mouse CHGB; rCHGB: rat CHGB; hCHGB: human CHGB, and zCHGB: zebrafish CHGB). (C) Secondary structure prediction (APSSP2, JPRED, and Polyview2D) of the hex fragment (CHGB521-591) which contains two helical segments, helix 2 and 3 (Hex 3) interspaced by a short loop. Helix 3 is the only long amphipathic helix in the MIF. (D) Alignment of the hex 3 fragments from different species. The alignment was performed using ClustalW and was manually adjusted. (E–G) Helical wheel representation of the CHGB helices 1–3 using an online server (http://rzlab.ucr.edu/scripts/wheel/wheel.cgi). (H) Light-scattering–based flux assay for EriC vesicles. EriC is a bacterial chloride/proton exchanger. (I) Vesicle floatation assay for CHGA vesicles. Gradient fractions were run in SDS–PAGE and the protein distribution was quantified in the plot under the gel. (J) Light-scattering–based flux assay from CHGA vesicles in different extra-vesicular buffers (pH 7.5 and 5.5 with or without 1.0 mM CaCl₂). The positive control of the CHGB vesicles (red trace) at pH 7.5 was showed for comparison.