Figure 3
From: The structural basis of nanobody unfolding reversibility and thermoresistance
Nanobody aggregation kinetics, structure and mechanism. (A) Monomer loss was monitored in centrifugation assays by separating aggregates from the soluble nanobody fraction at various time points. Measurements were performed in triplicate at the Tm value of each nanobody at a concentration of 32.7 µM. Dashed and solid lines represent single exponential fits. Fit parameters and Tm values are given in Supplementary Table 1. Orange-labeled nanobodies contain two disulfide bonds, blue labeled nanobodies exhibit one disulfide bond. (B) Distribution of aggregation onset temperatures Ts over different temperature regimes. (C) Relation of unfolding onset temperatures Ton and scattering onset temperatures Ts for the 64.6% of nanobodies aggregating within the unfolding transition of the heating-phase. The large majority of points lies above the diagonal, indicating that aggregation requires nanobody unfolding. (D–G) Electron micrographs of nanobody NbD1 aggregation at 32.7 µM: (D) Native protein; (E) fully aggregated protein after 30 min at 90 °C; (F,G) status after 35 min at the Tm value of NbD1 (65.4 °C).
