*3.1. Sample Preparation*

AZ (purity > 80%; MW = 14.6 kDa) was purchased from Sigma–Aldrich (St. Louis, MO, USA). The effective purity of the sample was checked by determining the ratio of spectral absorption at 630 nm and at 280 nm. AZ batches with a ratio value higher than 0.48 were used, with this indicating a good degree of purity [43]. AZ was dissolved in MilliQ water at a concentration of 200 μM. DBD (a.a 89–293; MW= 23 kDa) was purchased from GenScript (Piscataway, NJ, USA). DBD were dissolved in Phosphate Buffer Solution (PBS; 95.3% H2O, 3.8% NaCl, 0.1% di KCl, 0.7% Na2HPO4, 0.1% KH2PO4; pH = 7.4), reaching a final concentration of 40 μM. The DBD:AZ complex in PBS solution were prepared by mixing equimolar amounts of the components.

### *3.2. Raman Spectroscopy*

Raman measurements were carried out using a Super Labram confocal spectrometer (Horiba, France), equipped with several objectives, a diode-pumped solid-state laser (532 nm) and a spectrograph, with an 1800 g/mm grating allowing a resolution of 5 cm−1. Raman spectra were collected by means of a liquid nitrogen-cooled charged coupled device (CCD) (back illuminated; pixel format: 1024 × 128 detector) and in the back-scattering geometry in which a notch filter was used to reject the elastic contribution. All the experiments were performed using a laser power of 10 mW (4.4 mW on the sample) and a 50× objective with a numerical aperture NA = 0.6 (laser spot diameter reaching the sample was about 1 μm). A large confocal diaphragm (400 μm) and a slit of 200 μm were used to obtain a good Raman signal.

Protein drops (10–15 μL) were deposited onto an optical glass, and spectra were acquired on partially dried samples. Indeed, it was demonstrated that there are no significant differences between the Raman spectra of protein in solution and the corresponding drop coating deposition, in which the protein remains substantially hydrated and the secondary structure is largely preserved [44].

Each Raman spectrum was acquired at room temperature by averaging 10 scans of 10 s integration time. For each sample, twenty-five Raman spectra were collected from different regions of the drops. Raman data processing and analysis were performed with OPUS software version 6.5 (Bruker Optics, Ettlingen, Germany). All the spectra were normalized with respect to the phenylalanine (Phe) ring breathing band at 1002 cm−<sup>1</sup> due to its insensitivity to conformation or microenvironment [45], and the

fluorescence background was removed by applying a rubber band baseline correction [46]. Finally, the spectra used for the structural analysis were obtained by averaging five measurements to improve the spectral signal/noise ratio.
