 Solution

The S61/E62 and S75/Q76 pairs are especially important for the extra stimulation attributable to Munc18. Their replacement leaves basal lipid mixing broadly present but makes the red and blue traces much closer together. In contrast, replacement of R47/V48 or D57/Q58 retains strong stimulation. The S75/E78/T79 replacement retains a smaller enhancement, suggesting a partial contribution under these conditions.

Compare the Munc18-dependent increment with each construct's own baseline, rather than comparing red endpoints alone. These results identify functionally important regions; they do not prove that every altered side chain directly contacts Munc18. The original reconstitution experiments also support cognate-SNARE recognition.^[ref]