Fig. 4: Temperature-dependent NMR chemical shifts and temperature-dependent molecular dynamics.

a Experimental chemical shifts (relative to the APO state) of the five residues with the biggest change in temperature coefficient around 30 °C (between 292.9 K and 302.7 K, with slope displayed by dashed line) and around 50 °C (between 307.6 K and 322.4 K, with slope displayed by dotted line). Typical experimental error bars for the temperature coefficients are too small to be visualized (<1 ppb). b Experimental spectral overlay for the most prominent change in NMR chemical shifts (i.e, NH of fL63) at all temperatures under consideration. c Superimposition of the experimental temperature coefficient changes between 30 °C and 50 °C and the asymmetric dynamical perturbation contact network between all backbone NH moieties and the rest of the protein. The absolute variation between temperature coefficient around 30 °C and 50 °C are displayed in green spheres centered on the nitrogen atoms of N-H groups, with sphere sizes being proportional to the variation in the slope (ppb/K). Gray spheres refer to residues that could not be assigned unambiguously over the full temperature range in HisF, thus missing temperature coefficient values. In the perturbation network, blue and red edges represent a decrease and increase, respectively, of contacts upon an increase in temperature. Edge widths are proportional to the differences in number of contacts. d Evolution of the fL63-fR59 backbone hydrogen bond (defined as the distance between the backbone atoms fL63-N and fR59-O) over time in the apo30, apo50, and holo30. e–g Overlay of 100 configurations sampled (1 every ten ns) for the fL63 and fR59 residues along the apo30, holo30 and apo50 MD trajectories. The mean and standard deviation of the fL63-fR59 backbone hydrogen bond distance are also indicated. e–g Overlay of 100 fL63L and fR59 configurations sampled (1 every ten ns) along the apo30, holo30, and apo50 MD trajectories. While in apo30 fL63 and fR59 form a tight hydrogen bond, which is almost never present in holo30 (as highlighted by the long yellow arrow in g). apo50 represents an intermediate state between the two extremes, where the fL63 and fR59 become less stable upon temperature increase. Source data are provided as a Source Data file.