Chemical shift analysis
For each type of denaturant, the heteronuclear backbone resonances were assigned at three different denaturant concentrations (Fig. S3 in the SI). Moreover, the cold denatured state described in ref.27 was taken into account. The secondary chemical shifts of the protein in different denaturation conditions were rather similar to each other (Fig. S4).
To describe the transient structure in the denatured state under non-denaturing conditions, Cα, C’,15N and HN chemical shifts from individual titration series were extrapolated to the low intensity peaks observed at zero denaturant, as described in Fig. 3. As a result, in 16 cases the weak cross peaks observed at the position defined by the extrapolated values could be unambiguously assigned in the set of spectra recorded at physiological conditions at 25 ºC in the absence of any denaturant. For these 16 cases, the assignment by extrapolation was cross-checked and confirmed by 3D backbone spectra. The same extrapolation procedure was applied to all residues and the remaining plots are shown in supplementary Fig. S3 together with 9 of the identified cross peaks in the HSQCs.
In Fig. 4 we report the extrapolated secondary chemical shifts for the Cα, C’, N and HN backbone atoms averaged over the chemical shifts obtained from the four different extrapolations under different denaturing conditions. In these plots, we make use of intrinsic reference (i.e., the chemical shifts at highest denaturant concentration), although other choices gave similar results (see Fig. S5 in the SI). The error bars indicate the associated standard error and quantify the precision of the assignment under native conditions. Most residues displayed small errors compared to the average. A few discrepancies were observed for charged residues. The largest deviations were associated with the titration with acetic acid and were observed for three aspartic acids, D29, D30 and D60, and the single histidine, H69. Weaker effects were seen for four glutamates, E21, E34, E35 and E65. All the fits are displayed in Fig. S6 in the SI