Figure 3.

Physical interaction occurs between β₂ and the clamp-binding motif (CBM) of ε in the αεθ–β₂ complex. (A) β₂ binds to a peptide containing the ε_wt CBM. SPR binding isotherms (R/R_max) for the interaction of β₂ with immobilized decapeptides containing CBMs from ε_L (diamonds), ε_wt (circles), and ε_Q (squares) are shown; sensorgrams are in Supplementary Figure S3A. Fits to data using a 1:1 binding model (ε_L and ε_wt peptides) are shown as solid lines. The small responses with the ε_Q peptide at the highest [β₂] indicate K_D>2 mM (see Supplementary Figure S3A). (B) The CBM in ε_L is accessible to β₂. NanoESI mass spectra of 1 μM β₂ alone or with 20 μM ε_wt, ε_Q, or ε_L show that ε_L interacts more strongly with β₂ than does ε_wt. Proteins were in 140 mM NH₄OAc, and ions due to free monomeric ε (1700–3400 m/z) have been omitted for clarity. (C) Wild-type ε contacts β₂ in the αεθ–β₂ complex, shown by a shift in the αεθ: αεθβ₂ equilibrium (in excess β₂) with progressive increase in ε–β binding strength. NanoESI-MS of 2.8 μM β₂ with 1.8 μM purified αεθ cores in 140 mM NH₄OAc. Ions due to free β₂ (4000–5000 m/z) are not shown.