Additional File 5. File S5. PDB file of the obtained HEV Y-domain I-TASSER model (model 1) having... more Additional File 5. File S5. PDB file of the obtained HEV Y-domain I-TASSER model (model 1) having a C-score of -4.10.
Additional File 6. File S6. PDB file of the obtained HEV Y-domain I-TASSER model (model 5) having... more Additional File 6. File S6. PDB file of the obtained HEV Y-domain I-TASSER model (model 5) having a C-score of -4.25.
Additional File 2. Figure S2. The Ramachandran plot statistics of the generated 3D models of Y-do... more Additional File 2. Figure S2. The Ramachandran plot statistics of the generated 3D models of Y-domain of HEV (A) RaptorX; (B) Phyre2; (C) I-TASSER (model 1); and (D) I-TASSER (model 5).
Additional file 1. Figure S1. Maximum-likelihood phylogenetic tree of Y-domain protein gene seque... more Additional file 1. Figure S1. Maximum-likelihood phylogenetic tree of Y-domain protein gene sequences of HEV. This analysis involved 50 nucleotide sequences. Bootstrap values are represented by the numbers on nodes generated by 1000 replications.
Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in ch... more Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in children and infants. To date, there is no effective vaccine available against RSV. Heparan sulfate is a type of glycosaminoglycan that aids in the attachment of the RSV to the host cell membrane via the G protein. In the present study, the effect of amino acid substitution on the structure and stability of the ectodomain G protein was studied. Further, it was investigated whether mutation (K117A) in the CX3C motif of G protein alters the binding with heparan sulfate. The point mutation significantly affects the conformational stability of the G protein. The mutant protein showed a low binding affinity with heparan sulfate as compared to the wild-type G protein, as determined by fluorescence quenching, isothermal titration calorimetry (ITC), and molecular docking studies. The low binding affinity and decreased stability suggested that this mutation may play an important role in prevention ...
The global burden of disease caused by a respiratory syncytial virus (RSV) is becoming more widel... more The global burden of disease caused by a respiratory syncytial virus (RSV) is becoming more widely recognized in young children and adults. Heparan sulfate helps in attaching the virion through G protein with the host cell membrane. In this study, we examined the structural changes of ectodomain G protein (edG) in a wide pH range. The absorbance results revealed that protein maintains its tertiary structure at physiological and highly acidic and alkaline pH. However, visible aggregation of protein was observed in mild acidic pH. The intrinsic fluorescence study shows no significant change in the λmax except at pH 12.0. The ANS fluorescence of edG at pH 2.0 and 3.0 forms an acid-induced molten globule-like state. The denaturation transition curve monitored by fluorescence spectroscopy revealed that urea and GdmCl induced denaturation native (N) ↔ denatured (D) state follows a two-state process. The fluorescence quenching, molecular docking, and 50 ns simulation measurements suggested...
Beni-Suef University Journal of Basic and Applied Sciences, 2021
Background Hepatitis E virus (HEV) of the family Hepeviridae is a major causative agent of acute ... more Background Hepatitis E virus (HEV) of the family Hepeviridae is a major causative agent of acute hepatitis in developing countries. The Y-domain is derived from multi-domain non-structural polyprotein encoded by open reading frame 1 (ORF1). Previous studies have demonstrated the essentiality of Y-domain sequences in HEV life cycle; however, its function remains completely unexplored. The following study was thus conceptualized to examine the detailed computational investigation for the putative Y-domain to estimate its phylogenetic assessment, physiochemical properties, structural and functional characteristics using in silico analyses. Results The phylogenetic assessment of Y-domain with a vast range of hosts indicated that the protein was very well conserved throughout the course of evolution. The Y-domain was found to be unstable, hydrophilic and basic in nature with high thermostability value. Structural analysis of Y-domain revealed mixed α/β structural fold of the protein havi...
Hepatitis E virus (HEV) is a major causative agent of hepatitis E infections across the globe. Al... more Hepatitis E virus (HEV) is a major causative agent of hepatitis E infections across the globe. Although the essentiality of HEV nonstructural polyprotein (pORF1) putative Y-domain (Yd) has been established in viral pathogenesis, its structural-functional role remains elusive. The current research discusses the novel exploration on Yd protein expression, purification, biophysical characterization and structure-based docking analysis. The codon optimized synthetic gene and optimized expression parameters i.e., 5 h induction with 0.25 mM IPTG at 37 °C, resulted in efficient production of Yd protein (∼40 kDa) in E. coli BL21(DE3) cells. Majority of the recombinant Yd (rYd) protein expressed as inclusion bodies was solubilized in 0.5% N-lauroylsarcosine and purified using Ni-NTA chromatography. Circular dichroism (CD) and UV visible absorption spectroscopic studies on Yd revealed both secondary and tertiary structure stability in alkaline range (pH 8.0-10.0), suggesting correlation with its physiological activity. Thus, loss in structure at low pH perhaps play crucial role in cytoplasmic-membrane interaction. The biophysical data were in good agreement with in-silico structural analyses, which suggested mixed α/β fold, non-random and basic nature of Yd protein. Furthermore, due to Yd protein essentiality in HEV replication and pathogenesis, it was considered as a template for docking and drug-likeness analyses. The 3D modeling of Yd protein and structure-based screening and drug-likeness of inhibitory compounds, including established antiviral drugs led to the identification of top nine promising candidates. Nonetheless, in vitro studies on the predicted interaction of Yd with intracellular-membrane towards establishing replication-complexes as well as validations of the proposed therapeutic agents are warranted.
Respiratory syncytial virus (RSV) is a leading viral pathogen causing acute lower respiratory tra... more Respiratory syncytial virus (RSV) is a leading viral pathogen causing acute lower respiratory tract infection in children. The G protein of RSV is involved in attachment with the host cell. It is a neutralizing antigen and thus a vaccine candidate. Heparan sulfate is a type of glycosaminoglycan (GAG) present on the host cell membrane that is involved in attachment with the G protein of RSV. We describe a novel approach for efficient expression and purification of the ectodomain G protein in the prokaryotic system and its biophysical characterization. The native ectodomain G protein was purified using a two-step process by Ni-NTA and DEAE weak anion-exchange chromatography through the supernatant obtained after cell lysis. In addition, the denatured form of the protein was also purified from the solubilized inclusion bodies (IBs) by Ni-NTA affinity chromatography with a higher yield. Dynamic light scattering (DLS) was performed to confirm the homogeneity of the purified protein. The effect of pH on the stability and structure of the purified protein was studied by circular dichroism (CD), fluorescence, and absorbance spectroscopy techniques. Isothermal titration calorimetry (ITC) and microscale thermophoresis (MST) were exploited to demonstrate the interaction of heparan sulfate with the ectodomain G protein. The dynamic light scattering results showed that the purified protein was homogenic and had a well-folded native conformation. Biophysical characterization of the protein revealed that it was stable and had intact secondary and tertiary structures at pH 7.5. CD analysis revealed that the protein showed a loss in the secondary structure at pH values 5.5 and 3.5, while absorbance spectroscopy suggested a stable tertiary structure at pH values 7.5 and 5.5 with a probable aggregation pattern at pH 3.5. This loss in the structure of the ectodomain G protein at low pH can be correlated with its physiological activity. A slight change in pH might play a crucial role in host−pathogen interactions. The fluorescence intensity of the protein decreased on moving toward a lower pH with no spectral shift in emission maxima. In addition, isothermal titration calorimetry and microscale thermophoresis results showed strong binding affinity of the ectodomain G protein with heparan sulfate. The binding of heparan sulfate with protein was probably due to the electrostatic interaction of positively charged amino acid residues of the heparin-binding domain of the protein and the negatively charged group of GAGs. Future studies may involve the development of possible therapeutic agents interacting with the G protein and affecting the overall charge and pH that might hinder the host−pathogen interaction.
Aim: G glycoprotein ectodomain (Ge) of BA genotype of group B respiratory syncytial virus was exp... more Aim: G glycoprotein ectodomain (Ge) of BA genotype of group B respiratory syncytial virus was expressed and purified to achieve maximum yield of the protein. Materials & methods: We optimized different parameters like strains, temperature, inducer concentration and post induction time period for efficient protein expression in Escherichia coli. The protein was purified using affinity chromatography and confirmed by western blotting. Results: It was concluded that a 5-h induction with 0.75 mM isopropyl β-D-1-thiogalactopyranoside at 37°C in BL21(DE3) cells was the most favorable condition for maximal protein expression. The far-UV circular dichroism spectroscopy suggested that it is an α-helical protein. Conclusion: The purified Ge protein can be characterized by antigenic and biophysical methods in future studies, which will probably assist in vaccine development.
Additional File 5. File S5. PDB file of the obtained HEV Y-domain I-TASSER model (model 1) having... more Additional File 5. File S5. PDB file of the obtained HEV Y-domain I-TASSER model (model 1) having a C-score of -4.10.
Additional File 6. File S6. PDB file of the obtained HEV Y-domain I-TASSER model (model 5) having... more Additional File 6. File S6. PDB file of the obtained HEV Y-domain I-TASSER model (model 5) having a C-score of -4.25.
Additional File 2. Figure S2. The Ramachandran plot statistics of the generated 3D models of Y-do... more Additional File 2. Figure S2. The Ramachandran plot statistics of the generated 3D models of Y-domain of HEV (A) RaptorX; (B) Phyre2; (C) I-TASSER (model 1); and (D) I-TASSER (model 5).
Additional file 1. Figure S1. Maximum-likelihood phylogenetic tree of Y-domain protein gene seque... more Additional file 1. Figure S1. Maximum-likelihood phylogenetic tree of Y-domain protein gene sequences of HEV. This analysis involved 50 nucleotide sequences. Bootstrap values are represented by the numbers on nodes generated by 1000 replications.
Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in ch... more Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in children and infants. To date, there is no effective vaccine available against RSV. Heparan sulfate is a type of glycosaminoglycan that aids in the attachment of the RSV to the host cell membrane via the G protein. In the present study, the effect of amino acid substitution on the structure and stability of the ectodomain G protein was studied. Further, it was investigated whether mutation (K117A) in the CX3C motif of G protein alters the binding with heparan sulfate. The point mutation significantly affects the conformational stability of the G protein. The mutant protein showed a low binding affinity with heparan sulfate as compared to the wild-type G protein, as determined by fluorescence quenching, isothermal titration calorimetry (ITC), and molecular docking studies. The low binding affinity and decreased stability suggested that this mutation may play an important role in prevention ...
The global burden of disease caused by a respiratory syncytial virus (RSV) is becoming more widel... more The global burden of disease caused by a respiratory syncytial virus (RSV) is becoming more widely recognized in young children and adults. Heparan sulfate helps in attaching the virion through G protein with the host cell membrane. In this study, we examined the structural changes of ectodomain G protein (edG) in a wide pH range. The absorbance results revealed that protein maintains its tertiary structure at physiological and highly acidic and alkaline pH. However, visible aggregation of protein was observed in mild acidic pH. The intrinsic fluorescence study shows no significant change in the λmax except at pH 12.0. The ANS fluorescence of edG at pH 2.0 and 3.0 forms an acid-induced molten globule-like state. The denaturation transition curve monitored by fluorescence spectroscopy revealed that urea and GdmCl induced denaturation native (N) ↔ denatured (D) state follows a two-state process. The fluorescence quenching, molecular docking, and 50 ns simulation measurements suggested...
Beni-Suef University Journal of Basic and Applied Sciences, 2021
Background Hepatitis E virus (HEV) of the family Hepeviridae is a major causative agent of acute ... more Background Hepatitis E virus (HEV) of the family Hepeviridae is a major causative agent of acute hepatitis in developing countries. The Y-domain is derived from multi-domain non-structural polyprotein encoded by open reading frame 1 (ORF1). Previous studies have demonstrated the essentiality of Y-domain sequences in HEV life cycle; however, its function remains completely unexplored. The following study was thus conceptualized to examine the detailed computational investigation for the putative Y-domain to estimate its phylogenetic assessment, physiochemical properties, structural and functional characteristics using in silico analyses. Results The phylogenetic assessment of Y-domain with a vast range of hosts indicated that the protein was very well conserved throughout the course of evolution. The Y-domain was found to be unstable, hydrophilic and basic in nature with high thermostability value. Structural analysis of Y-domain revealed mixed α/β structural fold of the protein havi...
Hepatitis E virus (HEV) is a major causative agent of hepatitis E infections across the globe. Al... more Hepatitis E virus (HEV) is a major causative agent of hepatitis E infections across the globe. Although the essentiality of HEV nonstructural polyprotein (pORF1) putative Y-domain (Yd) has been established in viral pathogenesis, its structural-functional role remains elusive. The current research discusses the novel exploration on Yd protein expression, purification, biophysical characterization and structure-based docking analysis. The codon optimized synthetic gene and optimized expression parameters i.e., 5 h induction with 0.25 mM IPTG at 37 °C, resulted in efficient production of Yd protein (∼40 kDa) in E. coli BL21(DE3) cells. Majority of the recombinant Yd (rYd) protein expressed as inclusion bodies was solubilized in 0.5% N-lauroylsarcosine and purified using Ni-NTA chromatography. Circular dichroism (CD) and UV visible absorption spectroscopic studies on Yd revealed both secondary and tertiary structure stability in alkaline range (pH 8.0-10.0), suggesting correlation with its physiological activity. Thus, loss in structure at low pH perhaps play crucial role in cytoplasmic-membrane interaction. The biophysical data were in good agreement with in-silico structural analyses, which suggested mixed α/β fold, non-random and basic nature of Yd protein. Furthermore, due to Yd protein essentiality in HEV replication and pathogenesis, it was considered as a template for docking and drug-likeness analyses. The 3D modeling of Yd protein and structure-based screening and drug-likeness of inhibitory compounds, including established antiviral drugs led to the identification of top nine promising candidates. Nonetheless, in vitro studies on the predicted interaction of Yd with intracellular-membrane towards establishing replication-complexes as well as validations of the proposed therapeutic agents are warranted.
Respiratory syncytial virus (RSV) is a leading viral pathogen causing acute lower respiratory tra... more Respiratory syncytial virus (RSV) is a leading viral pathogen causing acute lower respiratory tract infection in children. The G protein of RSV is involved in attachment with the host cell. It is a neutralizing antigen and thus a vaccine candidate. Heparan sulfate is a type of glycosaminoglycan (GAG) present on the host cell membrane that is involved in attachment with the G protein of RSV. We describe a novel approach for efficient expression and purification of the ectodomain G protein in the prokaryotic system and its biophysical characterization. The native ectodomain G protein was purified using a two-step process by Ni-NTA and DEAE weak anion-exchange chromatography through the supernatant obtained after cell lysis. In addition, the denatured form of the protein was also purified from the solubilized inclusion bodies (IBs) by Ni-NTA affinity chromatography with a higher yield. Dynamic light scattering (DLS) was performed to confirm the homogeneity of the purified protein. The effect of pH on the stability and structure of the purified protein was studied by circular dichroism (CD), fluorescence, and absorbance spectroscopy techniques. Isothermal titration calorimetry (ITC) and microscale thermophoresis (MST) were exploited to demonstrate the interaction of heparan sulfate with the ectodomain G protein. The dynamic light scattering results showed that the purified protein was homogenic and had a well-folded native conformation. Biophysical characterization of the protein revealed that it was stable and had intact secondary and tertiary structures at pH 7.5. CD analysis revealed that the protein showed a loss in the secondary structure at pH values 5.5 and 3.5, while absorbance spectroscopy suggested a stable tertiary structure at pH values 7.5 and 5.5 with a probable aggregation pattern at pH 3.5. This loss in the structure of the ectodomain G protein at low pH can be correlated with its physiological activity. A slight change in pH might play a crucial role in host−pathogen interactions. The fluorescence intensity of the protein decreased on moving toward a lower pH with no spectral shift in emission maxima. In addition, isothermal titration calorimetry and microscale thermophoresis results showed strong binding affinity of the ectodomain G protein with heparan sulfate. The binding of heparan sulfate with protein was probably due to the electrostatic interaction of positively charged amino acid residues of the heparin-binding domain of the protein and the negatively charged group of GAGs. Future studies may involve the development of possible therapeutic agents interacting with the G protein and affecting the overall charge and pH that might hinder the host−pathogen interaction.
Aim: G glycoprotein ectodomain (Ge) of BA genotype of group B respiratory syncytial virus was exp... more Aim: G glycoprotein ectodomain (Ge) of BA genotype of group B respiratory syncytial virus was expressed and purified to achieve maximum yield of the protein. Materials & methods: We optimized different parameters like strains, temperature, inducer concentration and post induction time period for efficient protein expression in Escherichia coli. The protein was purified using affinity chromatography and confirmed by western blotting. Results: It was concluded that a 5-h induction with 0.75 mM isopropyl β-D-1-thiogalactopyranoside at 37°C in BL21(DE3) cells was the most favorable condition for maximal protein expression. The far-UV circular dichroism spectroscopy suggested that it is an α-helical protein. Conclusion: The purified Ge protein can be characterized by antigenic and biophysical methods in future studies, which will probably assist in vaccine development.
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