Extended Data Fig. 2: Purification and characterization of wFAPs.

a, Fluorescence emission spectrum of 1 µM HBC599 with or without 10 µM of the best early design, excited at 495 nm. The design weakly activated fluorescence of HBC599 over the buffer (Tris-buffered saline (TBS, 20 mM Tris, pH 7.4, 150 mM NaCl)). b, Three designs demonstrated fluorescence activation for HBC599. Relative fluorescence intensity was measured at 1 µM HBC599 with 1 µM of each designer protein. c, Normalized UV-vis absorbance spectra of wFAP1.1-HBC599 complex (orange) and free HBC599 (gray). d, Representative gel filtration chromatography and SDS-PAGE of wFAP0, wFAP1.1, wFAP1.2 and wFAP1.3. All four proteins eluted as monomers at similar volume on size-exclusion chromatography (SEC). For gel source data, see Supplementary Fig. 1. At least two independent experiments were performed, yielding consistent outcomes. e, Fluorescence-temperature curves of the wFAP1.1-HBC599 complex (red) and free HBC599 (black) in the temperature-induced dissociation experiment. Fluorescence gradually diminished to the background level as the temperature rose to 95 °C, and recovered to the original level when the sample cooled down. The dissociation midpoint of wFAP1.1 was approximately 60 °C. Data from three independent samples are presented as mean ± SD. f, Far-ultraviolet CD spectra of apo wFAP1.1 at 20 °C (grey line), 50 °C (red line), 95 °C (blue line) and cooled back to 20 °C (green line). g, Ligand specificity of wFAPs. 1 µM protein or buffer (TBS, pH 7.4) was mixed with 1 µM HBC for each combination, and fluorescence reading at the excitation and emission maxima for each condition was measured. Readings were normalized against that of the best protein-ligand combination.