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