Stability of hV3 is modulated by interaction of β7–β9 with the N-helix. (A) Representative data illustrating how the rate of increase in A₃₄₀ is faster at higher temperatures (left). The rate of aggregation proportionally increases with temperature. The data were fitted to an exponential function to obtain the rates of unfolding and aggregation (see Fig. S3). The rates were then fitted to a polynomial function to derive the E_act (right). Schematic illustrating how E_act is calculated (right). R = 1.987 kcal K⁻¹ mol⁻¹. (B) Global comparison of E_act derived from ME₂₁₅, HT₂₁₅, and A₃₄₀, using a heat map scheme. Mutants with the highest E_act are the most stabilizing and are on the right (green); destabilizing mutants show the lowest E_act and are on the left (red). In line with stability measurements in Fig. 5, the E_act also suggest that C2,36,65,229A variant is most stabilizing owing to the presence of C8, C122; and removal of C2, C8, or C122 destabilizes the barrel (see Fig. S4 for details). (C) Stabilizing α1-β7-β9 interaction is shown in green as a surface rendering on the hV3 structure. This stabilizing interaction is formed via interaction of C2, C8, and C122 (green spheres; other cysteines are in yellow).