Figure 2. Dimerization of EL222 variants both free in solution and bound to DNA.

(a) SEC-MALLS measurements of dark- and light-state AQTrip indicate the light-driven formation of a dimeric species. Under dark-state conditions AQTrip is largely monomeric (black) at the concentration used (140 µM). In contrast, at equivalent concentrations, AQTrip is a mixture of monomer and dimer in the light-state (red). (b) Mixtures of 120 µM light-treated AQTrip and 60 µM AN-45 DNA results in the formation of a more rapidly eluting species (blue) compared to light-state AQTrip alone (red) and DNA alone (black). The MW of the light-state DNA-bound EL222 complex is consistent with a 2:1 EL222: DNA ratio. (c) EMSA analysis of EL222 interactions with Clone-1 DNA¹⁶ (see Figure S1a for sequence), done in the presence of His₆-Gβ1-tagged and untagged WT EL222, confirms a 2:1 stoichiometry of EL222:DNA interactions. The presence of a mixed species (C2) compared to tagged (C3) and untagged (C1) protein/DNA complexes indicates that EL222 dimers interact with this 45 bp oligo. The lack of additional bands suggests monomeric or larger oligomeric states do not bind appreciably at the concentrations tested. (d) L120K is monomeric (MW=19 kDa) under dark-state conditions at 65 µM (black). Increasing the concentration to 155 µM (red) results in a small shift in both elution time and MW, up to a ~29 kDa species, consistent with a rapidly exchanging low affinity dimer^{27, 30}. For a, b, and d, each graph includes both UV absorbance measurements (top lines) and MALLS traces (bottom lines); MW measurements are provided from the midpoint of the MALLS traces shown.