(A) CD spectra of E, H, and reconstituted EH complex in 10 mM sodium phosphate, pH 7, 25 °C. (B) CD signal measured at 222nm as a function of increasing temperature (T-melt). For H, no cooperative loss of CD signal was observed, while E exhibited a cooperative transition around 25 °C (explored further in Figure 6). The melting curve of EH features two gradual transitions that were further studied by DSC due to the relatively high temperatures necessary to carry out the melt. (C) A comparison of the changes in heat capacity as a function of temperature for E, H and reconstituted EH. Thermograms were generated for each sample at a scan rate of 60 °C/hour from 10 °C to 115 °C, and buffer-buffer reference scans subtracted. The large exothermic signal acquired for E (red line) was due to sample aggregation/precipitation above 70 °C, and no significant changes in heat capacity could be seen for H (blue line). In contrast, large, endothermic peaks can be seen for the EH complex (green) above 60 °C. After baseline subtraction (D), data were best fit to a non-two-state model with three transitions (black, dotted lines).