MCL-1 gave a CD spectrum expected of a highly α-helical protein with double minima at 208 and 222 nm (Fig. ). Monitoring the minimum at 222 nm with increasing temperature range gave a thermal denaturation curve for MCL-1 with a midpoint Tm of 62 °C (Fig. & Table ), comparable to published data. The dimeric coiled coils CC-Di_S, CC-Di_E1 and CC-Di_E2 gave CD spectra consistent with αhelical coiled-coil assemblies (Fig. ). Across these peptides the relative thermal stability decreased (Table , Fig. ), i.e., as the length of the grafted binding sequence increased, coiledcoil stability decreased. CD data for the 1:1 mixture of CC-Di-A and CC-Di-B_S also confirmed a highly stable, α-helical assembly (Table & Fig. ). In contrast, the monomeric peptides NOXA-B (Fig. ) and Mono_E1 (Fig. ) showed minima around 198 nm and no unfolding transition (Fig and Fig. , respectively) consistent with disordered random coils, and, confirming that Mono_E1 does not form a coiled coil. CC-Tri_E1 and CC-Tet_E1 gave double minima at 208 and 222 nm (Fig. ), consistent with the high α-helical content, and unfolding curves with Tm ~60°C (broad) for CC-Tri_E1 and Tm >90 °C for CC-Tet_E1, comparable to the canonical de novo base CC-Tet coiled coil (Fig. ). 32 Coiled-coil peptide/MCL-1 complexes were prepared in a 1:1 manner of grafted peptide to protein; e.g., for homodimeric coiled coils CC-Di_E1 a 2:2 MCL-1/peptide sample were prepared, for CC-Tri_E1 a 3:3 MCL-1/ peptide sample and for the heterodimeric pair a 1:1:1 mix of CC-Di-A/CC-Di-B_S/MCL-1 were prepared. All peptide/MCL-1 samples gave strong α-helical CD spectra as expected (Fig. , S9 and S10a). Unfolding curves showed single transitions (Fig. . Thermal unfolding curves from the individual components were averaged to generate theoretical unfolding curves for the complex and predicted Tm(pre) values that assume no interaction between the components. (Note: The distinct transitions in the predicted curve for the mixture with CC-Di_E2 reflects the two very different unfolding profiles and Tm values for the individual components). Thus, comparing the theoretical and experimental curves indicates whether complex formation is stabilizing. In the presence of MCL-1, monomeric peptides NOXA-B (Fig. ) and Mono_E1 (Fig. ) gave unfolding curves with Tm values that were higher than predicted (Table ). All homodimeric coiled coil/MCL-1 complexes gave well-defined unfolding transitions with higher Tm values than the calculated averages, indicating thermal protection as observed previously for MCL-1 inhibitors. Most of the peptide-protein mixtures analysed, gave unfolding temperatures ~10 °C higher than calculated values. The small difference for CC-Di-A/CC-Di-B_S/MCL-1 (Fig. ) likely reflects the weaker inhibitory potency observed in the FA experiments. The data for CC-Di-E1 and CC-Di-E2 gave even higher values ~20 °C than predicted. Collectively, these data provide confirmation of direct coiled coil/MCL-1 interaction and reveal that complex formation is mutually stabilizing for the monomeric, dimeric and trimeric coiled coils; i.e., binding stabilizes both the coiled-coil assembly and the protein target against disassembly and thermal denaturation. The similarity in Tm for the monomeric, trimeric and tetrameric samples in the presence of MCL-1, may indicate interaction between CC-Tri_E1 or Tet_E1 and MCL-1 occurs at 1:1 stoichiometry (i.e. the oligomer dissociates in favor of MCL-1 binding, see later). Coiled Coils can Recruit Multiple Copies of the Target Protein. Complexes of the coiledcoil scaffolds with MCL-1 were evaluated by native mass spectrometry (Native MS) and analytical centrifugation (AUC) to assess oligomerization in the bound states.