Fig. 2: Biophysical characterization of LuxSit.
a, Coomassie-stained SDS–PAGE of purified recombinant LuxSit from E. coli (for gel source data, see Supplementary Fig. 1). b, Size-exclusion chromatography of purified LuxSit suggests monodispersed and monomeric properties. c, Far-ultraviolet CD spectra at 25 °C (black), 95 °C (red) and cooled back to 25 °C (green). Insert, CD melting curve of LuxSit at 220 nm. MRE, molar residue ellipticity. d, Luminescence emission spectra of DTZ in the presence (blue) and absence (green) of LuxSit. e, Structural alignment of the design model (blue) and AlphaFold2-predicted model (grey), which are in close agreement at both the backbone (left) and the side-chain (right) level. f–i, Site-saturation mutagenesis of substrate-interacting residues. Magnified views (left) of designed (blue) and AlphaFold2 (grey) models at the side-chain level, illustrating the designed enzyme–substrate interactions of Tyr14–His98 core HBNet (f), Asp18–Arg65 dyad (g), π-stacking (h) and hydrophobic packing (i) residues. Sequence profiles (right) are scaled by the activities of different sequence variants: (activity for the indicated amino acid)/(sum of activities over all tested amino acids at the indicated position). A96M and M110V substitutions with increased activity are highlighted in pink.
