Extended Data Fig. 3: Hallucinated Bid binders are stable and bind Bid peptide with high affinity.
From: De novo design of high-affinity binders of bioactive helical peptides
(a) 46 Hallucinated designs tested for initial experimental screening. (b) 4 designs were chosen for expression without Bid peptide. All expressed as monomeric proteins (assessed by preparative SEC) and were pure by SDS-PAGE (n = 1). (c) All Hallucinations could be pulled-down by biotinylated Bid immobilised on streptavidin magnetic beads. B = bound to bead, U = unbound, in supernatant. L = ladder (n = 1). (d) Bid is unstructured in isolation by circular dichroism (CD), whereas all Hallucinations were helical in isolation, as predicted from the Hallucinated structure. A 1:1 molar ratio of binder:Bid (Mix) produced greater helical signal than that predicted by the isolated spectra (No inter.) suggesting binding is inducing helix formation (n = 1). (e) Melting with CD showed that Hallucinations were thermostable, and binding to Bid increased thermostability (where measurable) (n = 1). All Hallucinated binders would remain folded, or refold after heating and cooling, in contrast to the natural binder Mcl-1 which precipitated in the process. (f) ITC showed that Hallucinations bound to Bid, with µM to nM Kds (n = 1). (g) FP measurements of designed Bid binders (n = 3).
