Figure 2.

SEC-MALS and SAXS analysis of apo-AfAgo and AfAgo-DNA complexes. (a) SEC-MALS analysis of WT AfAgo and dimerization mutant AfAgoΔ unbound to nucleic acids. The light scattering data (blue for WT AfAgo, green for AfAgoΔ) is shown along with the calculated M_w values (magenta for WT, black for mutant). The highest and lowest M_w values calculated for each protein are indicated. Theoretical M_w of WT AfAgo monomer is 50.8 kDa, theoretical M_w of AfAgoΔ monomer is 49.9 kDa. (b) SEC-MALS analysis of AfAgo-DNA and AfAgoΔ-DNA complexes. The UV absorption data of AfAgo-DNA (blue) and AfAgoΔ-DNA (green) is shown along with the M_w values of full complexes, the protein component, and the DNA component (magenta for AfAgo-DNA sample and black for AfAgoΔ-DNA sample, respectively). The theoretical M_w of a 2:2 WT AfAgo:DNA complex is 119 kDa (2 × 50.8 + 2 × 8.7 kDa), theoretical M_w of a 1:1 AfAgoΔ-DNA complex is 58.6 kDa (49.9 + 8.7 kDa). (c) SAXS data of WT AfAgo complex with MZ-1289 DNA (red points) and the dimerization mutant AfAgoΔ with MZ-1289 DNA (green points) are compared with the scattering curves generated from the “closed” dimer with dsRNA (PDB ID: 1ytu, black curves), “open” dimer (PDB ID: 2w42, blue curves) and AfAgo-DNA complex (PDB ID: 2w42, magenta curves) by CRYSOL. Corresponding AfAgo structures are shown in the second column superimposed with the dummy atom models generated using the SAXS data of the AfAgo complex with MZ-1289 oligoduplex.