FIGURE 3.

Purification and characterization of E_CT1 and E_CT1H_CT. A, overlay of E_CT1 and E_CT1H_CT elution profiles collected during passage of the samples over a16 × 500 mm Superdex 75 gel filtration column. Binding of H_CT to E_CT1 resulted in an elution volume shift from 52 to 60.5 ml. This corresponds to a change in estimated molecular mass (spherical protein) from 35 to 22 kDa, compared with actual molecular masses of 12. kDa and 15 kDa for E_CT1 and E_CT1H_CT, respectively. The change in elution volume suggests that H_CT binding effectively reduced the spherical volume of E_CT1, likely by out-competing homodimer formation. B, 15% SDS-PAGE gel of the E_CT1H_CT elution peak and individually purified components visualized by Coomassie staining. E_CT1 and H_CT were produced individually as fusion proteins and reconstituted as described under “Experimental Procedures.” C, CD wavelength scan of E_CT1 and E_CT1H_CT in 10 mm sodium phosphate (pH 7) at 25 °C. Although retaining a shape indicative of mixed secondary structure, E_CT1H_CT exhibits a greater molar ellipticity than E_CT1. Although this may be partially explained by the additional α-helical content contributed by H_CT, it is also likely that H_CT stabilizes E_CT1 fold. This is further supported by the increase in thermal stability observed for E_CT1H_CT versus E_CT1 alone (D) and additionally studied in Fig. 4.