Figure 5
From: The structural basis of nanobody unfolding reversibility and thermoresistance
Structural determinants of nanobody thermoresistance. (A) A second disulfide bond (SS) suppresses aggregation. Comparison of ΔTm shifts between dromedary-derived nanobodies with one and two disulfide bonds. ΔTm shifts were determined at 13.1 and 32.7 µM. The p-value refers to an unpaired t-test. (B) The CDR3 loop length contributes to nanobody thermostability. Relation of CDR3 loop lengths and Tm values measured at 13.1 µM. Average CDR3 lengths are 14 and 17 amino acids for nanobodies with one and two disulfide bonds, respectively. Dashed lines represent the linear regressions in the respective group-specific color; the 95% confidence interval is indicated by a colored band. Pearson correlation coefficients (r) are shown. One nanobody with two disulfide bonds was excluded from analysis (data point in brackets) because of its unusually short CDR3 length of 6 amino acids. (C) A long CDR3 loop suppresses aggregation in presence of a second disulfide bond. The correlation of CDR3 loop length and aggregation propensity was measured by ΔTm at 13.1 and 32.7 µM. Color codes and statistical parameters are as in panel A. (D) Nanobodies with a second disulfide bond are prone to lose their structure under reducing conditions. Relation of Ton values of nanobodies with one (blue) and two (orange) disulfide bonds in presence and absence of 25 mM TCEP, measured at 13.1 µM and a heating rate of 0.5 °C/min. A gray dashed line indicates a temperature of 37 °C.
