Figure 4.

SPR analysis of 2 and 4da. Concentrations refer to (A) c-Myc–Max(S) dimer or (B)–(E) small-molecule ligand. (A). Concentration-dependent binding of c-Myc–Max(S) heterodimers to a biotin-tagged E-box-containing double-stranded oligonucleotide immobilized to a streptavidin-coated biosensor chip. Binding of the oligonucleotide itself was associated with a response of 700–800 units. After resetting this value to baseline, equimolar concentrations of c-Myc and Max(S) were allowed to dimerize for 30 min and then passed over the biosensor chip. Note the concentration-dependent increase in response. (B). Prevention of heterodimerization by 2. The indicated concentrations of 2 were incubated with 20 nM of c-Myc for 20 min followed by the addition of 20 nM Max(S). Following an additional 30 min incubation, the mixture was applied to the E-Box-containing biosensor chip. (C). DNA binding by pre-formed c-Myc–Max(S) heterodimers is relatively resistant to 2. Pre-formed c-Myc–Max(S) heterodimers (20 nM of each protein) were incubated for 30 min. with the indicated concentrations of 2 and then applied to the E-Box-containing biosensor chip. (D). Disruption of DNA binding by 4da. The experiment was performed as described in (B) except that the indicated concentrations of 4da were incubated with c-Myc monomer prior to the addition of Max(S). (E). The experiment was performed as described in (D) except that pre-formed c-Myc–Max(S) heterodimers were incubated with the indicated concentrations of 4da. Blue bars indicate the period during which protein-compound mixtures were being injected. Note that the relative response in each case has been normalized to 100 to denote the maximal response for each set of curves in order to allow inter-group comparisons. Similar results for each panel were obtained on at least two additional occasions.