*3.3. Sample Preparation*

The protein stock solution was filtered with a Millipore filter disc of 0.22 µm. A broad range of different buffers was used to monitor the pH-dependent change in the structure of the edG. The buffer of pH 2.0 and 3.0 was prepared with 50 mM of glycine and adjusted the pH with HCl. For pH 4.0 and 5.0 buffer, we used 50 mM of acetate and adjusted the pH using acetic acid. The buffer of pH 6.0 and 7.0 was made 50 mM phosphate and adjusted the pH with NaH2PO4. For pH 8.0 and 9.0, we used 50 mM Tris and 100 mM NaCl and adjusted the pH using HCl. The buffer of pH 10.0, 11.0 and 12.0 was prepared with 50 mM of glycine and adjusted the pH with NaOH. Before taking the spectral measurements, the sample was incubated in a respected buffer for at least three hours to attain equilibrium.

We prepared the 8.7 M GdmCl stock solution and the freshly prepared 10.5 M stock solution of urea to examine the chemical-induced denaturation of the protein. The stock solution of both the chemicals was prepared in 25 mM Tris buffer (pH 8.0). For every measurement, protein concentration was fixed accordingly (0.3–0.6 mg mL−<sup>1</sup> ) with the required volume of buffer and urea/GdmCl. For more accuracy, every time, the concentration of the stock solution of protein was calculated using the molar absorbance coefficient (ε) of protein (8730 M−<sup>1</sup> cm−<sup>1</sup> ) at 280 nm. The calculated amount of protein and denaturant was taken and mixed correctly, and samples were incubated for at least 3–4 h at 25 ± 1 ◦C to ensure that the denaturation process was completed.
