2.1.3. Urea and GdmCl-Induced Denaturation

The G protein of RSV plays an important role in the host–pathogen interactions. To date, the 3D- structure of G protein is not known, and very limited information is available about its structural properties in solution. For instance, how does the G protein fold and unfold, how a protein behaves in a diverse solvent environment and how much protein is stable? The answer to these questions is still elusive. The behaviour of a protein in different solvent conditions gives information about protein folding and stability. Chemical-induced denaturation is a critical method to determine the structural stability of various proteins [46,47]. The stability and folding mechanism information will improve our knowledge of the behaviour of G protein in different biological conditions. We examined the urea and GdmCl induced denaturation by fluorescence spectroscopy to measure the stability of the edG.

**Figure 3.** ANS fluorescence spectra of edG in the pH range of 2.0–12.0 at 25 ◦C. The ANS was excited at 380 nm and recorded the emission spectra from 400–630 nm. The inset shows the ANS profile from pH 2.0–12.0, followed by observing changes in *F*<sup>490</sup> as a function of pH.

The protein stability can be measured by equilibrium unfolding studies in the presence of GdmCl or urea [48]. The edG has one tryptophan and five tyrosine residues which are often buried either partially or fully in the hydrophobic core of the folded protein. These aromatic amino acid residues of edG act as markers for the structural integrity of the protein. Figures 4A and 5A shows the fluorescence emission spectra of edG in the presence of increasing concentrations of urea and GdmCl, respectively. The decreased fluorescence spectra were observed with increasing concentrations of urea and GdmCl, with the shifting of λmax of tryptophan residues towards the longer wavelength (redshift). The native edG exhibit an emission maxima peak at 344 nm, and the λmax of the protein shifts to 356 nm at higher denaturant concentrations. From these observations, we confer that the tryptophan residues are shifted from nonpolar to the polar environment, as urea and GdmCl exposes the buried aromatic amino acid residues [49,50].

**Figure 4.** (**A**) Urea-induced denaturation of edG at pH 8.0 at 25 ◦C measured by intrinsic fluorescence studies. The emission spectra were recorded as a function of increasing urea concentrations (0.0−8.0 M). The protein was excited at 280 nm and recorded the 300−430 nm emission spectra. (**B**) Denaturation curve of edG (plot of *F*<sup>344</sup> as a function of [urea]). The inset in figure (**B**) shows the emission spectra of edG in the native and 8 M urea denatured state.

**Figure 5.** (**A**) GdmCl-induced denaturation of edG at pH 8.0 and 25 ◦C measured by intrinsic fluorescence studies. The emission spectra were recorded as a function of increasing concentration of GdmCl (0.0–6.0 M). The protein was excited at 280 nm and recorded the emission spectra from 300–430 nm. (**B**) Denaturation curve of edG (plot of *F*<sup>344</sup> as a function of [GdmCl]). The inset in figure (**B**) shows the emission spectra of edG in the native and 6 M GdmCl denatured state.

Our observation also suggests that as we increase the concentration of urea and GdmCl, unfolding of edG takes place, which exposes the buried tryptophan to more polar buffer conditions. The changes in the tryptophan microenvironment were monitored by *F*<sup>344</sup> (the emission wavelength at 344 nm) as a function of [urea] (Figure 4B) and [GdmCl] (Figure 5B). The plot of *F*<sup>344</sup> versus [urea] and [GdmCl] suggested that chemical-induced denaturation of edG occurred in a single step and followed a two-state transition mechanism. The transition curve shown in this figure were analyzed to estimate the stability parameter such as *m*, ∆G<sup>D</sup> <sup>0</sup> and C*<sup>m</sup>* from the denaturation curve using Equation (1). The values of these parameters are mentioned in Table 1. Hence, from these observations, we conclude that the tertiary structure of edG loses cooperatively without the participation of an intermediate state. The equilibrium unfolding transition induced by urea and GdmCl are not always equal; the difference might be due to the ionic character of GdmCl [51].


**Table 1.** Thermodynamic parameters obtained from urea and GdmCl-induced denaturation of edG at pH 8.0 and 25 ± 0.1 ◦C.

*2.2. Binding Interaction Studies of edG with Heparan Sulfate*

2.2.1. Fluorescence Quenching Measurements

The intrinsic fluorescence measurements of a protein are susceptible to its microenvironment, making it an important tool to investigate the formation of the complex between the ligand and protein [52,53]. The quenching mechanism of edG with heparan sulfate was studied to know the parameters such as binding constant (*K*), Stern–Volmer constant (*K*sv), and the number of binding sites (n). The binding constant of heparan sulfate interacting with protein was determined by exciting the protein at 280 nm, and the change in the fluorescence intensity was recorded in the range of 300–430 nm. The protein excitation at 295 nm was considered as fluorescence of only Tryptophan, while protein exited at 280 nm was considered as the excitation of phenylalanine, tyrosine, and tryptophan [54]. Figure 6A shows the fluorescence spectra of edG in the presence of an increasing concentration of heparan sulfate (0–50 µM). Heparan sulfate did not flourish alone, while protein gave a maxima peak at 344 nm in similar environmental conditions. A progressive decrease in

GdmCl N↔D 2.53 ± 0.20 1.66 ± 0.09 1.52 ± 0.07

the fluorescence spectra was observed with the addition of HS, indicating the formation of the complex between protein and ligand. The quenching data was analyzed using the Stern–Volmer Equation (2) to calculate the Stern–Volmer constant (*K*sv). The Stern–Volmer plots of protein quenching in the presence of HS is shown in Figure 6B. The *K*sv value was determined from the Equation by plotting the fluorescence intensity ratio *F0/F* for different concentrations of HS. The value of the bimolecular quenching constant (*Kq*) was obtained using Equation (3) and further confirmed the mode of quenching. In the presence of HS, the decrease in fluorescence intensity was analyzed by the modified Stern–Volmer Equation (4). Figure 6C shows the fitted experimental data based on the double log relation with the intercept of the plot providing the binding constant. The binding constant (*K*) value was found to be 3.98 <sup>×</sup> <sup>10</sup><sup>6</sup> , which confirmed that HS has a high binding affinity to edG. The binding parameters of the edG-HS system calculated from the fluorescence quenching are given in Table 2. Interestingly, our previous binding studies by microscale thermophoresis (MST) and isothermal titration calorimetry (ITC) [30] complement the results of the fluorescence binding study. However, various reports had mentioned the difference in the thermodynamic parameters obtained from ITC and fluorescence quenching. This difference is due to fluorescence quenching, as it measures only the local changes around the fluorophore, whereas the ITC and MST measure the global changes in the thermodynamic properties [55].

**Figure 6.** Fluorescence binding studies of the edG with heparan sulfate (HS) at pH 8.0 at 25 ◦C. The protein was excited at 280 nm and recorded the emission spectra from 300−430 nm. (**A**) Fluorescence emission spectra of protein with increasing concentration (0−50 µM) of HS. (**B**) Stern−Volmer plot for quenching of edG−HS complex. (**C**) Modified Stern−Volmer (double-log relation) plot of the edG−HS complex.

**Table 2.** Binding parameters of the edG with heparan sulphate were obtained from fluorescence quenching studies at pH 8.0 and 25 ◦C (298 K).


2.2.2. Absorbance Binding Measurements

The absorbance spectra of edG give a characteristic peak at 278 nm due to the presence of aromatic residues. The change in the spectra indicates the interaction of the ligand to protein [56]. A gradual decrease in the absorption spectra of edG was observed with an

increasing concentration of heparan sulfate (HS). The protein spectra in the absence of ligand (HS) give a peak at 278 nm. As we increase the ligand concentration 0–50 µM, a significant decrease in the spectrum has been observed with some scattering in the range of 340–320 nm (Figure 7). However, the 278 nm peak shifted towards a shorter wavelength (blueshifts) with increasing ligand concentration, confirming that the aromatic amino residues of the protein are exposed to a more polar environment [57].

**Figure 7.** Absorbance binding measurements of the edG with heparan sulfate (HS) at pH 8.0 and 25 ◦C. The spectra were recorded in the range of 340–240 nm with increasing concentration (0–50 µM) of HS. The inset shows the 278 nm peak and shifting of spectra towards shorter wavelength (blue shift) with increasing concentration of heparan sulfate.
