Figure 2.

Comparison of measured melting curves using the normalized data sets at pH = 7. (a) Schematic overview of the applied biophysical approaches in characterization of the thermal unfolding of ^Δ1–14Ex4-Tc5b variants. (b) Normalized molar ellipticity values at 222 nm [Θ_222nm]_MR and (c) folded fraction values derived from deconvolution are obtained by far-UV CD spectroscopy. (d) Normalized sums of secondary chemical shift values describing helicity (SCS_helix) and (e) Tc fold compactness (SCS_cage) determined by NMR. In all four cases (b–e), larger values indicate a higher degree of structural order. Note that the NMR-derived data (4–48 °C) cover only a fraction of the temperature range used to record the CD spectra (5–85 °C). Each data set has been normalized to the respective data point corresponding to the ^Δ1–14Ex4-Tc5b_QR measured at the lowest temperature. This standardizes the scale and units of the results obtained by different techniques, facilitating the comparison of the recorded melting curves. These reference data points are considered as 1. (f) Normalization of the measured data points allowed us to determine the temperatures at which the variants reach the same structural order as the reference ^Δ1–14Ex4-Tc5b_QR at 4 °C (horizontal red dotted line, b–e). (g) Comparison of the fold descriptors at the highest temperature, where all four values could still be determined (vertical black line, b–e).