Figure 3.

Thermal unfolding of MtGimα, MtGimβ, and MtGimC monitored by CD at 222 nm. (a) Thermal unfolding of isolated MtGimα and MtGimβ subunits displayed as the fractions folded and fitted to a two-state transition, as described previously (26). Subsequent cooling of the heated samples resulted in curves of identical shape and corresponded to similar T_m values (not shown). (b) Thermal unfolding of 1.2 μM MtGimC shown as the temperature dependence of the mean residue weight ellipticity (Θ_MRW) at 222 nm. Changing the KCl concentration from 0 M (1), 0.2 M (2), 0.4 M (3), and 0.6 M (4) shifts the T_m values for the denaturation of MtGimC to higher temperatures. Whereas the high-temperature transition can be fitted with a two-state model, the transition midpoint of the distorted low-temperature transition was defined by approximating lines corresponding to the temperature dependences of Θ_MRW of the folded complex (F) and the intermediate (I), as shown in the plot. The temperature at which the observed Θ_MRW intersects a third line (M) representing the midpoint between F and I was used as the denaturation temperature. The distorted shape and T_m values did not change by using Tris- or ammonium acetate-based buffer systems or slower heating rates. The low-temperature transition is irreversible, as cooling produces a refolding curve characteristic of a cooperative process and, relative to the unfolding transition, a decreased T_m value (48.3 ± 1.5°C). (c) Concentration dependence of the transition midpoints of the MtGimC and MtGimα melting curves in the absence of salt. The T_m values of both the low- and high-temperature transition T_m values of MtGimC are concentration dependent (▾, low-temperature transition; ▿, high-temperature transition). The concentration dependence of the high-temperature transition corresponds to that of MtGimα, □.