Figure 3.

Protein stability of S-crystallin and its mutants by heat denaturation (a,b) and chemical denaturation (c,d). Plots of the relative CD signal from the ellipticity at 222 nm as a function of the temperature for the wild-type (magenta) and various S-crystallin mutants, the L100F/D101N double mutant (green), the ∆loop/L100F/D101N double mutant (orange), and the L100F/D101N/M104V/Q108F quadruple mutant (blue) without (a) or with (b) 1 mM GSH. The thermal stability of GST-σ (red) without or with 1 mM GSH was also measured. The protein concentration was 7.2 μΜ. The results were fitted in order to calculate their T_m of each mutant. which are shown in Table 2. (c,d) Denaturant-induced light scattering of S-crystallin (c) and its mutants (d). The aggregation traces of S-crystallin in 3.5 M urea without or with GSH at 1–2 mM were observed by light-scattering at 340 nm. The protein concentration was 10 μM. The assays were repeated twice to ensure reproducibility.