Stability of hV3 is modulated by specific cysteine residues. (A) Schematic illustrating the thermal parameters measured using far-UV CD from changes in ME₂₁₅ or absorbance at 340 nm (A₃₄₀) normalized between 0 and 1. The profile illustrates T_m-start, T_m, and T_m-end, used here as indicators of protein stability. Here, T_m-start = temperature where unfolding or aggregation is nucleated; T_m = the temperature where 50% unfolding or aggregation is achieved; and T_m-end = the temperature where unfolding or aggregation is completed. (B) Thermal denaturation profiles of C2,8A (red) and C2,36,65,229A (green) variants normalized between 0 and 1, highlighting the dependence of the measured thermal parameters on the mutation. Here, C2,36,65,229A protein exhibits high value of T_m-start and T_m, denoting high stability. (C) Comparison of T_m-start and T_m derived from ME₂₁₅ measurements for the most (C2,36,65,229A and C8,122A in green) and least (C2,8A in red) stable mutants of hV3, obtained from the global comparison of T_m-start and T_m from ME₂₁₅ and A₃₄₀ (see Fig. S2 C). Data for WT is shown in gray for comparison only. Error bars represent SD derived from a minimum of three independent experiments. Statistical significance (***, P < 0.001; **, P < 0.01; *, P < 0.05) was derived using t test (unpaired). (D) Schematic highlighting the importance of C2/8-C122 interactions for hV3 stability. When both C2 and C8 are mutated (red spheres) the protein is least stable (left); mutating or retaining both C8 and C122A as a pair stabilizes the barrel (right).