Figure 3.

Analysis of Max_22–113 monomer–dimer equilibrium and kinetics and [Max_22–113]₂⋅Ebox₂₂ association kinetics. (A) Max_22–113 monomer–dimer equilibrium analyzed by CD. (B) Max_22–113 monomer–dimer kinetics analyzed by CD and stopped-flow fluorescence. A 50-μM solution of Max_22–113 was diluted 50-fold into PBS buffer at 25°C, and the negative ellipticity at 222 nm was monitored as a function of time. (Inset) Max_22–105^SFlu monomer–dimer relaxation kinetics monitored by stopped-flow fluorescence polarization within the first 200 sec of the reaction. A solution of 5 μM Max_22–105^SFlu was diluted 10-fold into PBS buffer at 25°C, and the fluorescence anisotropy was monitored as a function of time. (C) Association of 1 nM (○) and 2 nM (□) Max_22–113 with 50 pM Ebox₂₂ at 25°C. Each point represented the average of at least three independent experiments. Error bars shown represent the SD. Similar association kinetics were observed with a 135-bp DNA fragment containing a single Ebox site (CACGTG). Simulations of binding of Ebox₂₂ by Max_22–113 via the monomer (solid lines) and dimer (dashed lines) pathways are shown.