Figure 4.

Comparison of the side chain interaction network modulating the 3₁₀ helices. The molecular basis behind the observed thermostability differences of Tc variants lies in distinct interactions between residues Q24, W25, D/E/Q28, S33, and R35 (depicted in ball-and-stick representation) of the 3₁₀ helix motifs for the following variants: (a) ^Δ1–14Ex4-Tc5b_CC, (b) ^Δ1–14Ex4-Tc5b, (c) ^Δ1–14Ex4-Tc5b_ER, and (d) ^Δ1–14Ex4-Tc5b_QR. In the interaction networks (lower left corner), hexagons stand for the key residues, with connecting edges showing the number of the assigned NOE cross peaks at two temperatures: 4 °C (dark blue) and 26 °C (red). In water, the NMR-based assignment of some functional groups with exchanging protons, such as the guanidino group of Arg or the hydroxyl group of Ser, is challenging. Therefore, the detection of some of these signals is indeed informative and underlines the robustness of the Tc fold (for NOE data, see Table S1). The network of these side chain interactions defines the geometry of the 3₁₀ helices, shown as the NMR ensembles depicted in the bottom right corner of each panel. The decrease in the “edge” numbers is an indication of the degree of resistance to thermal unfolding of the 3₁₀ helices. The protons of the observed D28-R35 salt bridge and the OH proton of S33 are both detected in the case of ^Δ1–14Ex4-Tc5b_CC and ^Δ1–14Ex4-Tc5b, showing the extent of their burial within the hydrophobic core of the miniprotein. The mere presence of such groups in the NMR spectra provides valuable information about the 3D structure. Table (e) summarizes the different interactions that contribute to the 3₁₀ helix and Tc fold compactness.