Extended Data Fig. 5: Scanning alanine mutagenesis of the POM1 paratope.
From: A conformational switch controlling the toxicity of the prion protein
Extended Data Figure 5. (a) Intermolecular contacts between human PrPC120–230 and POM1 Fab variable heavy chain (magenta, left panel), and POM1 Fab variable light chain (green, right panel) as determined by Baral et al., 20128. Reproduced with permission of the International Union of Crystallography from doi:10.1107/S0907444912037328. (b) Schematic representation of a single-chain fragment of wild-type POM. The mutated residues are indicated as stick on the cartoon structure of POM1, color coded as in Supplementary Table 1b. The CDR loops are shown from the perspective of the antigen. (c) Scheme of competition FRET assay to assess the KD of various pomologs. In the absence of competing antibody, FRET occurs due to proximity of allophycocyanin (APC)-labeled holo-POM3 and europium (Eu3+)-labeled POM1 (left panel). Because of liquid-phase competition, addition of unlabeled pomologs leads to a decrease in FRET signal (right panel). The calculation of binding constants from FRET is detailed in the methods section. (d) The binding constants measured by SPR and by FRET were in good agreement, Spearman r = 0.77, p=0.0074, 95% CI 0.30–0.94) with the exception of hcW33A, whose binding on SPR was too weak to be precisely measured.
