Figure 4: Epitope mapping of wild type and D76N β₂m in their complexes with Nb24.

(a) Structural map of the complexation induced shifts () for the backbone amide ¹⁵N-¹H NMR peak frequencies in wild type β₂m (pale gray) and D76N β₂m (pale green) upon interaction with Nb24 nanobody. For wild type β₂m and D76N β₂m, the average  ± σ values of 0.08 ± 0.06 and 0.08 ± 0.06 ppm were obtained, respectively. NH locations with larger than one standard deviation from the average shift are indicated by spheres, with larger deviations by increasing color darkness. The two classes of chemical shift perturbations here presented indicate surface residues (orange-brown grading), and internal residues (cyan grading). For both β₂m species, the effects of Nb24 binding are observed at the apical loops AB and EF and the C-terminal segment that represent a conformational epitope moiety. A further perturbation involves several consecutive residues of CD loop identifying a sequential epitope portion. Allosteric conformational effects involve internal residues at the end of strand B and, within strands C and F. (b) Elements of secondary structure in β₂m and D76N β₂m where residue locations are shown in yellow, orange and red when the corresponding backbone amide, upon Nb24 binding, exhibits a value larger than (_av + σ), (_av + 2σ) and (_av + 3σ), respectively.