**1. Introduction**

p53 is an important tumor suppressor protein working as a central hub in a complex interaction network in which it regulates numerous cellular processes, including cell cycle progression, apoptosis induction, and DNA repair [1,2]. p53 is a member of the important class of intrinsically disordered proteins (IDPs), possessing both structured and disordered domains under physiological conditions and different conformations coexisting in solution [3]. Such a structural plasticity confers to IDP an extremely high conformational adaptability, allowing them to act according to functional modes not achievable by ordered proteins, with these properties having been recently exploited to develop engineered protein and peptide drugs [4–6].

p53 is a tetrameric protein composed of four identical subunits and acts as a transcription factor. Each monomer of p53 consists of an N-terminal transactivation domain (NTD), a C-terminal domain (CTD), and a core DNA-binding domain (DBD) [7–10] The presence of unstructured portions allows p53 to adopt widely different conformations, which are at the basis of a vast repertoire of available interactions to different biological partners [11]. Among them, Azurin (AZ), a copper-containing electron-transfer anticancer protein secreted by *Pseudomonas aeruginosa* bacteria, has demonstrated the ability to specifically bind to p53, leading both to its stabilization and to an intracellular level increase both in vitro and in vivo [12–17]. Therefore, the formation of the p53-AZ complex has opened new perspectives in cancer treatment, such as the development of an AZ-derived anticancer peptide [18].

Keeping in mind the crucial role of AZ in assisting the oncosuppressive function of p53, in our group, we investigated the interaction between p53 and AZ at the single molecule level by Atomic Force Microscopy (AFM) and Atomic Force Spectroscopy (AFS) and by computational approaches [12,19–21]. These studies have provided information about the interaction kinetics between p53 or its DBD and AZ, obtaining also some relevant insights on the possible binding sites [21]. However, no experimental evidences on possible structural alterations of p53 upon its binding to AZ are so far available [3]. In this respect, Raman spectroscopy represents a suitable approach to extract information about the secondary structure of proteins as well as to probe their conformational heterogeneity, including IDPs [22]. Indeed, we have previously applied such a technique to investigate the structure and the conformational heterogeneity of wild-type and mutants p53 and, also, of the AZ-derived anticancer p28 peptide, even in different environmental conditions [18,23,24].

In the present work, we have employed a Raman-based approach to investigate if and how the native conformation of DBD is modified by its interaction with AZ. To such an aim, we have focused on an accurate inspection of the Fermi doublets relative to Tyrosine (830 and 850 cm−1; Tyr) and of Tryptophan peaks (1340 and 1360 cm<sup>−</sup>1), with these Raman signals having been recognized as suitable diagnostic markers of protein side chain environment [25,26]. Additionally, we have investigated the Amide I Raman band (1600–1700 cm−1), of which the deconvolution has demonstrated to be particularly effective in both extracting conformational information (α-helix, β-sheet, and random coil motifs) and which is a reliable reporter on the structural heterogeneity of proteins [22,27–32]. The Raman spectra have also been analyzed by applying principal component analysis (PCA), which performs a dimensionality reduction of the spectra, allowing a revelation of the differences between the complex Raman spectra of the samples and helping to understand the principal factors affecting the spectral variation [33].

The combination of these approaches has put into evidence the occurrence of structural changes within p53DBD upon its interaction with AZ. In particular, passing from isolated DBD to DBD bound to AZ, we found a variation in Tyrosine (Tyr) and Tryptophan (Trp) residues hydrophobicity and an increase of the DBD secondary structure concomitantly with a significant reduction of the conformational heterogeneity. The observed changes in both the structure and conformational heterogeneity of DBD strongly support the ability of AZ to modulate the DBD structure, and this, in turn, may result in a stabilization of the oncosuppressive function of p53.
