Figure 6. Quaternary interactions of E1G1, C1, a1_NT, and H.

(A) Model of the quaternary subunit assembly. Dotted arrows indicate weak and solid arrows (black) strong binding. (B) Gel filtration profile of H/C1/a1_NT/E1G1 mixture (red) in comparison to H (purple), C1 (blue), a1_NT (yellow), and E1G1 (green) monomers. (C) SDS-PAGE analysis of the eluted fractions from gel filtration chromatography. Border colors indicate samples corresponding to the color scheme used in 6B. “C” indicates control proteins. (D) Panel X: Basic native polyacrylamide gel electrophoresis analysis of the H/C1/a1_NT/E1G1 mixture. A 3∶2∶1∶1 molar ratio of E1G1:a1_NT:C1:H proteins was prepared and incubated on ice for 1 h (lane 5). Bands corresponding to one molar amounts of E1G1, C1, a1_NT, and H proteins are visible in lanes 1, 2, 3, and 4, respectively. Panel Y: SDS-PAGE (12% gel) analysis of the intense band eluted from the native gel in panel X (lane 5), suggesting the presence of C1, E1 and G1 in this complex. An unbound a1_NT band was observed at the expected position. (E) SDS-PAGE of the eluted proteins from the His-tag pulldown experiment. Lane 1, fraction eluted using buffer B; lane 2, subunits bound with His-tagged H subunit eluted using buffer C.