Extended Data Fig. 4: Biochemical and structural characterizations of designs BB1–4.
a, Results of experimental characterization of the nonfunctional designs (BB1–4). Reproducibility is described in the Methods. †E value is calculated by BLAST, the non-redundant protein database. b, Far-ultraviolet CD spectra of designs BB2 and BB3 at 25 °C. c, SEC–MALS analysis showed a major monomer peak for BB1 and a major tetramer peak for BB2. d, Variants of BB1 with residues of the tryptophan corner and glycine kinks mutated to alanine were purified and sized. SEC traces are superimposed on the SEC trace of wild-type BB1 (WT). The mutations of all residues of the tryptophan corner eliminate the monomeric peak. Most of the glycine kink mutations negatively affect the monomeric species. The exceptions are Gly53 and Gly55, which are next to each other on the fourth strand. One glycine kink per strand might be sufficient to introduce enough negative twist to remove strain in the β-barrel. e–f, Deviations between BB1 design model and crystal structure. e, One of the three bottom turns of the crystal structure (grey) deviates from the design model (magenta) and forms additional crystal contacts (indicated by a dashed circle). f, Three phenylalanine side chains have different rotameric states. In the crystal structure, Phe41 interacts with Gly53 (which shows the most backbone deviation between the crystal structure and the design) to form an aromatic rescue motif53. It is likely that the discrepancies in the Phe rotamers reflect a scoring and sampling challenge to accurately capture such aromatic rescue; molecular dynamics simulation starting from the crystal structure (cyan) was also unable to recover the correct Phe41–Gly53 interaction. g, Biophysical properties (absorbance or fluorescence spectra, quantum yield and binding affinity) of mFAP1 and mFAP2 in complex with DFHBI. Mean values from three biological replicates were used for the nonlinear regression to determine the KD. The error estimates are the standard deviation from the fitting calculation. *λabs is peak absorbance wavelength, λex is peak excitation wavelength and λem is peak emission wavelength. †Absolute quantum yield is measured with an integrating sphere; relative quantum yield is measured using acridine yellow and fluorescein as the standards. ‡Previously reported value26. §Taken from previously published work54. ||Taken from previously published work31.
