**4. Conclusions**

The structural and conformational changes in the DBD region of the intrinsically disordered protein p53 upon interacting with the anticancer blue copper protein AZ were investigated by applying Raman spectroscopy. A careful inspection of the Raman spectra combined with a PCA analysis on the Fermi doublets of the Raman markers corresponding to the tyrosine and tryptophan residues allowed us to monitor the changes in their microenvironment as induced by the formation of a complex between DBD and AZ. Interestingly, we found a direct involvement of DBDTrp146 in the complex formation, as suggested by other experimental investigations. Additionally, a deconvolution of the Amide I band, remarkably sensitive to the α-helix, β-sheets, and random coil structures, allowed us to quantify the main secondary structural motifs of the DBD and its changes as induced upon binding to AZ. We found that DBD undergoes a slight increase of the β-conformation, with a concomitant lowering of its disordered portions as well as of its conformational heterogeneity. These findings are in agreement with our previous computational results and suggest that the binding of AZ to some unstructured motifs of DBD can restrain their flexibility. Collectively, the observed modulation the DBD structure when bound to AZ may represent a ground for understanding the molecular mechanisms of the AZ anticancer activity and could provide some hints for designing other molecules for p53-targeted therapies. Finally, we would remark that our Raman-based approach can be applied to investigate the structural changes of other biomolecules undergoing specific complex formation in order also to elucidate the molecular mechanisms which regulate their biological functions.

**Author Contributions:** S.S. data curation, formal analysis, investigation, and writing—original draft preparation; S.C. and A.R.B. writing—review and editing; S.C. and A.R.B. conceptualization supervision.

**Funding:** This research was funded by the Italian Association for Cancer Research (AIRC) Grant IG15866 to S.C.

**Conflicts of Interest:** The authors declare no conflict of interest.
