Figure 3.

Analysis of the temperature-dependent Ex4-Tc5b chimera structural ensembles calculated using ¹H–¹H NOESY-derived constraints. (a) Number of assigned ¹H–¹H NOESY cross peaks at each temperature. (b) Aromatic region of the ¹H–¹H NOESY spectra of ^Δ1–14Ex4-Tc5b (black) and ^Δ1–14Ex4-Tc5b_QR (teal). Cross peaks corresponding to the side chains of Tyr8 and Trp11 were analyzed to determine the interaction network within the hydrophobic core of Tc. The abundance of the NOESY signals in this region provides valuable insight into the compactness of the Tc motif. An increase in the number of signals introduces additional restraints in the structural calculations, resulting in a more precisely defined and well-folded structure ensemble. (c–f) Structural ensembles (indicating their respective PDB entries) of Ex4-Tc5b derivatives as temperature elevates (left to right) as well as their all-atom RMSD values plotted along the sequence. Each ensemble consists of the 10 lowest energy structures calculated, shown in a semitransparent visualization, along with their average structure overlaid. Long-range NOESY cross peaks (≥i + 5), which are critical for determining the tertiary Tc motif, are shown adjacent to the structure ensembles.