Extended Data Fig. 6: Biochemical and structural characterization of design b10, b32 and b11.

a, Size-exclusion chromatogram of His6-tagged b10 and b32 after Ni-NTA affinity purification. The monodispersed peaks of absorbance at 280 nm of b10 and b32 (cyan and lavender, respectively) have an elution volume compatible with the monomeric β-barrel (14 kDa), on the basis of their relative position to the protein standard peaks (dashed line). Biological replicates were performed with similar observation: n = 4 for b10, n = 5 for b32. b, Comparison of the ligand-binding pocket in the b10 design model (middle, grey) with the crystal structure (left, cyan). The side chain disagreements are highlighted with a dashed black circle on the right panel. c, d, The designed disulfide bond as a stabilizing mechanism. SEC curves of His6-tagged b11 (purple line) and b38 (dark yellow line) were overlaid to show the appearance of a monomer peak for b11 (the same standard as in a was applied here). A disulfide bond connecting the N-terminal helix to a β-strand (Q1C and M59C, circled in d) and four mutations of neighbouring residues were introduced into design b38 (dark yellow) to make design b11 (purple). Biological replicates were performed with similar observation: n = 3 for b38, n = 5 for b11. e, Far-ultraviolet CD spectra of b10, b32 and b11. Left, spectra at different temperatures within one heating–cooling cycle. Right, thermal melting curves (the CD signal of b10 was monitored at 220 nm; b32 and b11 at 226 nm). b11 probably forms an amyloid-like beta structure at 95 °C (left, bottom row) with a negative peak around 226 nm⁵⁶ and refolds back after cooling to 25 °C. The thermal stability of b11 decreases when the disulfide was reduced with 1 mM tris(2-carboxyethyl) phosphine (TCEP) (right, bottom). Measurements were performed once for each design (n = 1). f, Fluorescence emission spectra of b32, b11 and b11L5F in complex with DFHBI. With 200 μM proteins, b32, b11 and b11L5F can activate 10 μM DFHBI fluorescence by 8-, 12- and 18-fold, respectively. Two biological replicates were performed with similar results. g, The residues designed to interact with DFHBI contribute to b11 and b32 activity. Single or double knockouts of hydrogen-bonding residues (Y71, S23, N17 and T95) and a hydrophobic-packing residue (M15) showed decreased fluorescence intensity at 500 nm in comparison with the wild-type b11 or b32 (WT). Mutants were purified once for activity measurement. h, i, Re-designed five-residue fifth turn in b11L5F. The original bulge-containing ‘AAG’ β-turn in b11 (Extended Data Fig. 3b) was redesigned into a five-residue turn. b11L5F was detected by yeast surface display and flow cytometry (i and Supplementary Data). Yeast cells displaying b11 and b11L5F showed an increased 520-nm fluorescence signal (excited by 488-nm laser, i). Three biological replicates were performed with similar observation.