**1. Introduction**

Respiratory syncytial virus (RSV) is a lower respiratory tract pathogen that causes pneumonia and bronchiolitis in children, especially those younger than five years of age. It can also affect the elderly and immunocompromised individuals [1–3]. It infects about 70% of children less than one-year-old, 2–3% of whom are hospitalized. By the age of 2, almost all the children get infected due to RSV [4]. At present, there is no licensed vaccine or any therapeutics available against RSV. The only available preventive measure is the injection of monoclonal antibodies cocktail (palivizumab) specific to the fusion glycoprotein, which may decrease the severity of disease and hospitalization of infants [5]. In the developmental race for RSV vaccines, one of the major concerns is whether to incorporate the G glycoprotein in the subunit of live vaccines or not. Some scientists claim that the presence of G protein causes a proinflammatory response, as seen in the formalin-inactivated vaccine. On the other hand, some claim that it induces neutralization antibodies, which may be helpful in protection against RSV [6,7]. However, some monoclonal antibodies (mAbs) which target the G protein neutralize the infection of RSV on human airway epithelial cells and reduce the viral load and disease titer in an animal model [8–10]. Additionally, anti-G protein mAbs re-establish the Th1/Th2 cytokine level and suppress the pulmonary inflammation, mucus production and pro-inflammatory cytokines [11–14].

RSV is a negative-sense, single-stranded RNA virus enclosed in an envelope. The 15.2 kb genome of the RSV encodes the 11 proteins, out of which three are structural proteins SH (small hydrophobic), G (attachment glycoprotein), and F (fusion glycoprotein) that are embedded in the lipid envelope and play a significant role in viral entry, attachment, and fusion respectively. The attachment G glycoprotein is a type II membrane protein with an N-terminal cytoplasmic domain (1–36 aa), transmembrane domain (37–66 aa), and extracellular ectodomain (67–298 aa). The extracellular domain of the G protein comprises a central conserved domain (CCD), composed of 13 amino acids (164–176 aa) which are conserved among all the RSV isolates [15]. The cluster of four cysteine residues is present at 173, 176, 182, and 186 positions joined by two disulfide bonds formed between the Cys173–186 and Cys176–182 residues. The third and fourth cysteine residues form the CX3C motif (182–186 aa), which helps in the attachment of RSV to the susceptible human airway epithelium cell with the interaction of CX3CR1, a chemokine receptor [16,17]. A positively charged heparin-binding domain (184–198 aa) of the G protein also facilitates the attachment of the RSV via cell surface glycosaminoglycans (GAGs) [18,19]. Heparan sulfate (HS) is a type of GAGs molecule which exhibit various biological activities, most of which facilitate attachment with protein [20]. The HS molecules are present in almost all types of mammalian cells and act as a coreceptor for a number of viruses [21]. Recently, studies have suggested that HS act as a coreceptor for the Spike protein of SARS CoV-2, which makes this interaction an attractive target for SARS CoV-2 infection [22].

Protein is an essential biological macromolecule with enormous importance in every physiochemical process. The native or folded form of proteins is necessary to perform their biological function [23]. Conditions responsible for a protein to remain in the native conformation in the solution are pH, temperature, ionic strength, cofactors, and chaperon proteins [24,25]. In some cases, the stability of the protein is also controlled by the addition of osmolytes viz., trehalose and proline that stabilize the native conformation of the protein [26,27]. However, urea and guanidium chloride (GdmCl) are the major chemical denaturant used for proteins [28]. The stability of the protein depends on the noncovalent interaction, which can be disrupted using these denaturants [29]. Several studies are available which determine protein stability by studying denaturation curves [30,31]. Due to the unavailability of the crystal structure of G protein, limited information is available about its biophysical properties. Some studies reported that the G protein behaves differently in different environmental conditions such as pH and temperature. Currently, there is no information available that reports the folding and unfolding behaviour of G protein and determine its structural and functional stability. In this report, we, for the first time, determine the structural conformation change of ectodomain edG in a wide range of pH and determine the protein stability in the presence of denaturant (urea and GdmCl). To understand how different environmental conditions affect the structure and function of edG, it is important to understand the basic mechanism of host–pathogen interactions.

Here, we expressed and purified the ectodomain G protein (edG). The purified protein was used to determine the structural and conformational stability in different environmental conditions (pH, urea, GdmCl). Further, we reported the binding interaction of heparan sulphate with edG by fluorescence quenching, molecular docking, and molecular dynamics (MD) simulation studies.
