Fig. 5: Analysis of the unfolded states of SNase by DOPA2 cross-linking.

a The crystal structure of SNase (PDB code: 1JOO⁷¹). b The unfolding transition of SNase in different concentrations of urea monitored by fluorescence emission at 325 nm. In the y-axis, a. u. stands for arbitrary units. c Circular dichroism spectra in far-UV region measured for SNase in the presence of urea. d Experimental workflow of cross-linking SNase in different concentrations of urea using DOPA2. e 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 SNase 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). f The cross-links identified in different concentrations of urea were mapped on the crystal structure of RNase A (PDB code: 1JOO⁷¹). Cluster A is framed in blue (23 pairs), Cluster B in orange (64 pairs), and Cluster C in green (7 pairs). A red line denotes that the distance between two cross-linked residues exceeds the maximal cross-linking distance of DOPA2, and a black denotes that it does not. We performed two independent cross-linking experiments for each sample, and each 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, c, e) are provided as a Source Data file.