*3.3. Analysis of the Raman Spectra*

The secondary structure content of isolated DBD and AZ was quantified through a deconvolution procedure of the Amide I Raman bands by using three pseudo-Voigt profiles. The model parameters were optimized with the Levenberg–Marquardt minimization algorithm (LMA), and the goodness of the fit was assessed by the reduced chi-square value. The AZ curves as extracted from the fit were used in the DBD:AZ complex analysis, under the hypothesis that the AZ secondary structure does not change upon the interaction [38]. The three pseudo-Voigt profiles were centered at 1650–1656, 1664–1670, and 1680 cm−<sup>1</sup> and assigned to α-helix, β-strand, and random coil conformations, respectively, as validated on other IDPs [22,23,27]. In each fitting analysis, additional peaks had to be included in the band-fitting protocol to account for aromatic residue modes (1550, 1580, 1604, and 1615 cm−1) and for disordered structure and/or vibronic coupling (1637 cm−1) not baseline separated from Amide I features [22]. The errors relative to secondary structure percentages were evaluated by repeating the curve-fitting procedure on five different spectra and the accuracy associated with the determined secondary structure content was about 10% for each sample.

In order to improve the performance of deconvolution analysis, we performed a dimensionality reduction of the Raman spectra based on principal component analysis (PCA) [33]. The PCA transforms the original data set into a new data set with transformed variables (principal components) that are linear combinations of the original variables. The principal components were arranged in a swat that the variability of the original data set was contained in descending order in the first principal components. PCA was applied to the isolated DBD and AZ molecules and to the DBD:AZ complex (number of spectra n = 75) in three different spectral regions: (i) Fermi doublets relative to Tyr (830 and 850 cm<sup>−</sup>1), satisfactorily described by a number of components N= 79; (ii) Fermi doublets relative to Trp (1340 and 1360 cm<sup>−</sup>1) described by N = 79; and (iii) Amide I Raman band (1600–1700 cm<sup>−</sup>1) described by N = 77. The number of components of the correlation matrix to be considered was defined as the number required to explain at least 80% of the total variance. STATISTICA 7.0 software (StatSoft Inc., Tulsa, OK, USA, 2004) was used for all the analyses.
