Fig. 4: Allosteric state switching is cooperative.
From: De novo design of allosterically switchable protein assemblies
a, Theoretical distributions of peptide-bound ring states (rings bound to 0, 1, 2, 3 or 4 peptides) for a tetrameric ring across a peptide-titration series from 0 to 100% fractional saturation of binding sites for a non-cooperative binder (top) and an infinitely cooperative binder (bottom). Left and right, modelled structures of sr312 in the Xm and Yn states. b, SEC of 5 μM sr312 in absence of peptide (top), with 5 μM peptide (middle) and with 10 μM peptide (bottom). Protein (230 nm) and GFP (473 nm) absorbance (Abs) are shown in black and green, respectively. Conc., concentration. c,d, MP on fractions corresponding to earlier (c) and later (d) peaks in b, middle subpanel. Icon shows oligomeric species that matches the estimation of mass. Vertical lines indicate expected masses of fully bound, unbound and partially liganded species. P refers to the number of peptides bound to the ring. e, The 15 most populated 2D nsEM classes from nsEM on the mixture shown in b, middle subpanel. f, Characterization of sr312’s cooperativity. MP on samples of 3 μM sr312 incubated with 0, 2, 3, 5, 7 and 8 μM GFP-tagged peptide. Vertical lines represent expected masses of GFP-bound and apo species. g, MP on samples of 3 μM of sr312_locked incubated with 0, 1, 2.5, 4 and 5 μM GFP-tagged peptide. h, Models of intermediate oligomeric states occupied as sr508 binds to ligand, with X or Y conformational states of component hinges denoted below each stage. i, Characterization of sr508’s cooperativity. MP on samples of 3 μM sr508 incubated with 0, 0.5, 1, 3, 4 and 6 μM GFP-tagged peptide. Vertical lines represent expected masses of GFP-bound and apo species. j, Measurement of affinity and cooperativity in sr312 (top) and sr508 (bottom). Solid line, ligand-binding curves estimated from MP across a range of peptide concentrations, with sr312 and sr508 held at a constant concentration of 2 μM and 250 nM, respectively (Methods). Each point represents the mean of three measurements, with error bars representing standard deviation. Dissociation constant (Kd) and Hill coefficient (n) estimated by nonlinear regression are shown. Dotted line, equivalent ligand-binding curve with the same Kd and a Hill coefficient of 1. Icons indicate the nature of oligomeric change in each case.
