Fig. 7: Analysis of the unfolded states of RNase A by DOPA2 cross-linking.

a Experimental workflow of cross-linking RNase A in different concentrations of denaturants (urea or GdnHCl) using DOPA2. b, c Circular dichroism spectra in far-UV region measured for RNase A in the presence of denaturants. d The changes of spectral counts for each identified cross-linked residue pair (normalized across the six conditions, shown as Z-scores on the left axis) and the mean residue ellipticity (MRE) of RNase A monitored by CD at 222 nm (on the right axis) in different concentrations of urea. The residue pairs were classified into three clusters by K-means (Cluster A, Cluster B, and Cluster C). e As in (b), but in different concentrations of GdnHCl. f The cross-links identified in different concentrations of urea were mapped on the crystal structure of RNase A (PDB code: 6ETK⁶⁸). Cluster A in blue frame (11 pairs); Cluster B in orange frame (17 pairs); and Cluster C in magenta frame (3 pairs). The black lines denote that the distance of cross-linked residue pairs is within the restraints of cross-linkers, while the red lines denote that the distance of cross-linked residue pairs is out of the restraints of cross-linkers. The four pairs of disulfide bonds are colored dark blue. We performed two independent cross-linking experiments for each sample. Each cross-linking reaction was analyzed twice by LC-MS/MS. Cross-linking residue pairs were filtered by requiring FDR < 0.01 at the spectra level, E-value < 1 × 10⁻⁸, and spectral counts > 3. Source data for (b–e) are provided as a Source Data file.