*2.1. Raman Analysis of AZ and DBD*

Figure 1 shows the Raman spectra of AZ and DBD in the 600–1725 cm−<sup>1</sup> frequency range. The spectra display a complex set of bands arising from the modes of the aromatic amino acids (Tyr, Trp, and Phenylalanine (Phe) and of the peptide backbone, consistent with the typical Raman spectra of proteins [27,34]. The assignments of the main peaks are summarized in Table 1 [27].


**Table 1.** Typical proteins' Raman vibrational modes (Raman cm<sup>−</sup>1) and related assignments.


**Table 1.** *Cont.*

**Figure 1.** Raman spectra (600–1730 cm<sup>−</sup>1) with excitation at 532 nm of Azurin (AZ; blue) and DNA-binding domain (DBD; magenta) in Phosphate Buffer Solution (PBS): The principal proteins' vibrational modes are marked. Spectra were normalized in the all spectrum frequency region and baseline corrected for a better visualization.

Among the main Raman markers, we focused our attention on the Raman peaks of Tyr and Trp residues, which allow the extraction of information on protein side-chain local environment and on the Raman band of Amide I, which provides a diagnostic of the protein secondary structure.

Concerning the Tyr residues, the ratio IY = I850/I830 between the intensity of doublet peaks at 850 and 830 cm−<sup>1</sup> is related to the donor or acceptor role of the Tyr phenoxyl group. Specifically, a low IY value (around 0.3) indicates the phenolic hydroxyl (OH) group acting as a strong hydrogen bond donor, as occurring for buried tyrosine residues. As the IY value increases (until 2.5), the phenolic oxygen becomes a stronger hydrogen bond acceptor, while a largely enhanced value (IY > 6.7) represents a non-hydrogen-bonded state [25,26]. Experimental results on isolated AZ reveal an IY value of 0.38 <sup>±</sup> 0.07, representative of a buried environment for its two Tyr residues (Tyr<sup>72</sup> and Tyr108) in agreement with the X-ray structure of AZ, in which Tyr<sup>72</sup> belongs to the peripheral α-helix region with a moderate solvent accessibility and Tyr108 is practically inaccessible to solvent (see Figure 2A) [35].

**Figure 2.** Three-dimensional structures of (**A**) AZ (PDB code: 4AZU) and (**B**) DBD (PDB code: 2XWR): The active site of AZ and the zinc-finger of the DBD are shown as yellow ball and stick models. The aromatic residues are Tyr (green) and Trp (orange). The OH groups in Tyr residues are marked in red.

Isolated DBD exhibits an IY ratio of 1.37 ± 0.16, indicating a predominant exposition to the solvent surfaces of the eight Tyr residues. From X-ray structure, the phenolic OH groups of Tyr<sup>103</sup> and Tyr107 are highly oriented towards the solvent (see Figure 2B) [7]. Moreover, Tyr<sup>126</sup> and Tyr205, located at a crucial protein region interfacing with the DNA, show moderate accessibility, similar to that of Tyr<sup>220</sup> located on the surface of the protein [7,8]. Finally, the remaining Tyr163, Tyr234, and Tyr236 are almost inaccessible to the solvent [8]. Therefore, our results are consistent with the X-ray data endorsing the DBD–Tyr high solvent exposition.

Further information about the side chains can be achieved by analyzing the Fermi doublet bands of Trp residues at 1340 and 1360 cm−1, which are reporters of the hydrophobicity/hydrophilicity neighboring the Trp indole ring [32]. In particular, an intensity ratio IW = I1360/I1340 smaller than 1.0 reflects a hydrophilic environment, while a ratio greater than 1.0 indicates a hydrophobic one [9,32].

For AZ, we found an IW ratio of 1.54 ± 0.10, indicative of a buried and solvent inaccessible environment for the lone Trp residue. This is in accordance with the AZ X-ray data, showing that the Trp48 is deeply embedded in a highly hydrophobic core and surrounded by a closely packed β barrel structure (Figure 2A) [35].

We found for DBD an IW ratio of 0.68 ± 0.10, which implies, on average, a moderate hydrophilic environment for its Trp<sup>91</sup> and Trp146. The latter is positioned in a hydrophobic side chain and oriented towards the solvent, while the former is located at the N-terminus of DBD and displays a high solvent accessibility, as it comes out from the X-ray data (Figure 2B) [36]. However, Trp91 has been shown to be crucially involved in the packing process of DBD through interaction with the Arg174 residue, which reduces its solvent exposure [36]. Therefore, our data suggest that both Trp residues in DBD globally experience a hydrophilic environment.

Information on protein secondary structure can be extracted by the Amide I band (1600–1700 cm<sup>−</sup>1), mainly arising from C=O stretching and the combination of the C–N stretching, the Cα-C–N bending, and the N–H in-plane bending modes of peptide group. Such a band is usually used as a marker for secondary structure components. In particular, when Amide I band is centered at 1655 cm−1, it indicates a prevailing α-helix conformational arrangement, while a shift of this band peak toward 1670 cm−<sup>1</sup> is indicative of β-sheet conformation [22]. On the other hand, an analysis of the Amide I shape means an appropriate deconvolution strategy allows for the quantification of the percentage content of secondary structure components present in the protein [22,28].

Specifically, the Amide I band of AZ emerges at about 1670 cm−<sup>1</sup> (Figure 3A), suggesting a predominant β-sheet conformation [37]. The curve-fitting procedure points out β-sheet conformations predominant for 60%, while the α-helices and random coils account for about 22% and 18%, respectively. The obtained AZ secondary structure is agreement with that determined by X-ray diffraction for the crystals of AZ. Indeed, the major AZ components are β strands and turns (≈69%), which form two sheets arranged in a Greek key motif and with a minor contribution from a rigid α-helix (about 31%), conferring to AZ a low level of flexibility and structural disorder (see Figure 3A) [21,35,38].

**Figure 3.** The Amide I band of AZ (**A**) and DBD (**B**) in PBS: The percentage of secondary structure for these proteins has been estimated from the relative area of deconvoluted bands of this spectral region of which the fitting parameters are reported in Table 2.

**Table 2.** Assignments, relative central frequency (Raman cm<sup>−</sup>1), and integrated intensities (Area %) of the main Amide I band components (α-helix, β-sheet, or random coil) for AZ, DBD, and DBD:AZ complex obtained by a fitting procedure. χ2 = 0.002 for all curve fitting analysis.


Concerning DBD (Figure 3B), the band corresponding to the β-structures provides 46% of the total, while those related to α-helix and to random coils have 25% and 29%, respectively. These results indicate that DBD is characterized by a partially ordered structure, combined with the presence of significant disordered regions. Additionally, the results confirm those reported in our recent study on different sample batches of DBD (aminoacids 81–300), from which a content of 27% and 50% for α-helical and β conformations, respectively, have been estimated [23]. Moreover, these data are in agreement with X-ray data indicating a 30% of β-arrangement with an 18% of α-structures (see Figure 3B) [36]. The DBD propensity to adopt a predominant β-conformation is actually related to the large presence in its sequence of hydrophobic residues, such as Cysteine (Cys), Trp, and Leucine (Leu), generally promoting an ordered structure [39].
