Figure 5: Nb24 rescues partially unfolded D76N β₂m.

(a) Overlay of ¹⁵N-¹H HSQC spectral regions obtained from different D76N β₂m samples, namely 57 μM with peak doubling because of partial unfolding onset (blue contours) and 75 μM with native conformation and no sign of heterogeneity (red contours). Both samples were prepared in 25 mM aqueous phosphate (pH* 7.2) and observed within a few days from preparation at 25 °C. The assignments for most of the doubled signals are reported. (b) The same D76N β₂m solutions, as in panel A, were prepared with addition of Nb24 (see Methods), at analogous nanobody/protein concentration ratios, i.e. 114 μM/57 μM (blue contours) and 165 μM/75 μM (red contours), respectively. Peak doubling proved completely removed and the coincidence between the spectra of D76N β₂m bound to Nb24 demonstrates that this nanobody rescues partial unfolding of the highly amyloidogenic β₂m mutant. (c) Structural distribution of the D76N β₂m residues involved in the early partial unfolding documented in panel A. The apical region (in pale brown) closely matches the epitope recognized by Nb24, whereas the D strand fragment involvement (in cyan) is an extension of the conformational heterogeneity from the preceding CD loop.