2.1.2. Fluorescence Measurements

Fluorescence studies provide information about the protein's tertiary structure. The aromatic amino acid residue is extremely sensitive to its local environment, and it is responsible for a protein's intrinsic fluorescence measurements. A characteristic redshift in λmax is indicative of the increased interaction of aromatic amino acid residue upon the unfolding of the protein in the solvent [36]. The edG contains five tyrosine and one tryptophan residue, which enables us to perform the intrinsic fluorescence to observe the effect of pH on the tertiary structure of the protein. The changes in λmax were plotted as a function of pH to determine the alteration in the microenvironment of the buried aromatic amino acid of the protein. The changes in intrinsic fluorescence of edG at the wide range of pH are shown in Figure 2.

**Figure 1.** Absorbance spectra of edG at different pH values (2.0−12.0) at 25 ◦C. The spectra were measured in the range of 340−240 nm. The spectrum at pH 8.0 is considered as a control. The inset shows the denaturation profile of edG from pH 2.0−12.0 followed by observing changes in ∆ε<sup>278</sup> as a function of pH.

**Figure 2.** Fluorescence emission spectra of edG in the pH range of 2.0–12.0 at 25 ◦C. The protein was excited at 280 nm and recorded the 300–400 nm emission spectra. The inset shows the denaturation profile of edG from pH 2.0–12.0, followed by changes in emission maxima (λmax) as a function of pH.

The native conformation of edG at pH 8.0 shows λmax at 344 nm. The emission maxima show no spectral shift, as we are moving from acidic pH to alkaline pH condition (except pH 12.0), attributed that the microenvironment of aromatic residues was significantly disturbed. The decrease in fluorescence intensity was observed as we moved from the physiological pH to acid pH values. The decrease in intensity might be due to the protonation of acidic amino acids or water molecules surrounding the aromatic amino acid residues. Similarly, a substantial decrease in fluorescence intensity was observed as we moved from the physiological pH to alkaline pH values (pH 9.0–12.0). This indicates the deprotonation of essential amino acids present around the intrinsic fluorophore that leads to fluorescence quenching. The deprotonation/protonation of amino acid side chains may lead to disruption of charge in the local environment by interrupting internal salt bridges and electrostatic interactions that are present in the native conformation of the protein [37]. The plot of λmax versus pH shows no significant change in the emission maxima of edG from pH 2.0–11.0. However, a redshift of 5 nm in λmax was observed at pH 12.0 (see inset of Figure 2). The characteristic redshift of 5 nm in emission maxima is indicative of the increased solvent interaction of aromatic amino acid residue upon unfolding of the protein. From our fluorescence measurements, we concluded that the tertiary structure of edG remains similar at pH 8.0–9.0. However, destabilization of charges on protein surface leads to the disruption of the electrostatic interaction at acidic and basic pH values.

The changes in protein structure under various environmental conditions often lead to the exposure of hydrophobic patches normally buried in the native state. The ANS is an extrinsic dye that shows binding with partially unfolded protein in which tertiary structure was distorted and secondary structure of the protein retained. The high binding affinity of ANS to these confirmations confirms the presence of pre molten globule (PMG) or molten globule (MG) state [38,39]. The binding of ANS with hydrophobic patches leads to an increase in fluorescence intensity along with the blue shift in emission maxima [40,41]. Figure 3 shows the change in fluorescence intensity in the presence of ANS at different pH values. We found that the ANS fluorescence intensity decreases as we move towards the basic pH values. The ANS fluorescence intensity of protein at pH 2.0 and 3.0 was much higher than the native state (pH 8.0) with shifting of emission maxima towards the shorter wavelength (blue shift). This might be due to the exposure of buried hydrophobic clusters in non-native states of protein populated at pH 2.0 and 3.0. The above-mentioned features are the well-established characteristics of the molten globule (MG) state (partially folded intermediate state of a protein) that is induced at mild denaturing conditions [42,43]. Hence, the non-native state of edG at pH 2.0 and 3.0 is considered an acid-induced molten globulelike state. With native and alkaline conditions, no significant difference in fluorescence intensity was seen (see inset of Figure 3). This resulted from the solvent inaccessibility of buried hydrophobic clusters, which prevents the ANS binding [44,45].
