Understanding the mechanism of hV3 unfolding and aggregation. (A) Schematic of how possible aggregation mechanisms affect the measured T_m. A lower value of T_m-ME215 than T_m-A340 suggests the transition of native protein to unfolded species followed by the formation of structured aggregates (Mechanism 1). On the other hand, T_m-ME215 greater than T_m-A340 indicates that the folded protein directly forms structured aggregates during the unfolding process; hence, the unfolded species is not directly detected (Mechanism 2). (B) Histogram comparing T_m-ME215 and T_m-A340. A rainbow color pattern is used to represent the progressive lowering in cysteine content from hV3 WT (dark blue; left extreme) to the Cys-less mutant C0 (dark green; right extreme). Here, WT and all mutants show T_m-A340 < T_m-ME215, indicating hV3 oligomerization and aggregation before complete unfolding or loss in secondary structure (see Fig. S7 for details). The only deviation is observed in C2,8A (see text for discussion). Overall, hV3 follows Mechanism 2, where protein unfolding and aggregation are coupled.