Figure 7. YscU_C suppressor mutant stabilities and dissociation kinetics.

(A) Thermal induced unfolding of YscU_C and suppressor mutants. Dissociation temperatures (T _diss) of single suppressor mutants at pH 7.4 were quantified with the CD signal at 220 nm and a scan rate of 1°C/min; YscU_C (black), A268F (blue), Y287G (green), and V292T (red). All suppressor mutants are destabilized compared to wild-type YscU_C. T _diss values are summarized in Table 3. (B), (C) Dissociation kinetics quantified as dissociation life-times (τ_diss) of YscU_C (black) and V292T (red) at pH 7.4 followed with NMR-spectroscopy at (B) 37°C and (C) 30°C, respectively. Primary NMR data for (B) is shown in Figure S6. Solid lines correspond to fits of the experimental data to single exponential decays. (D) Time dependent GST-pulldown experiments show the dissociation of wild-type YscU_C and the suppressor mutant A268F at 30°C and 37°C after varying incubation times. The suppressor mutant A268F displayed pronounced dissociation of YscU_CC-His₆ at 37°C and moderate dissociation at 30°C; wild-type YscU_C displayed no dissociation of YscU_CC-His₆ at 37°C or at 30°C over the observed time period. Note! Dissociation of YscU_CC is manifested as disappearance of YscU_CC-His₆ over time since the dissociation is irreversible and YscU_CC-His₆ cannot bind itself to the used resin.