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.
Chikungunya fever is an arboviral infection caused by the Chikungunya virus (CHIKV) and is transmitted by Aedes mosquito. The envelope protein (E2) of Chikungunya virus is involved in attachment of virion with the host cell. The present study was conceptualized to determine the structure of E2 protein of CHIKV and to identify the potential viral entry inhibitors. The secondary and tertiary structure of E2 protein was determined using bioinformatics tools. The mutational analysis of the E2 protein suggested that mutations may stabilize or de-stabilize the structure which may affect the structure-function relationship. In silico screening of various compounds from different databases identified two lead molecules i.e. phenothiazine and bafilomycin. Molecular docking and MD simulation studies of the E2 protein and compound complexes was carried out. This analysis revealed that bafilomycin has high docking score and thus high binding affinity with E2 protein suggesting stable protein-ligand interaction. Further, MD simulations suggested that both the compounds were stabilizing E2 protein. Thus, bafilomycin and phenothiazine may be considered as the lead compounds in terms of potential entry inhibitor for CHIKV. Further, these results should be confirmed by comprehensive cell culture, cytotoxic assays and animal experiments. Certain derivatives of phenothiazines can also be explored in future studies for entry inhibitors against CHIKV. The present investigation thus provides insight into protein structural dynamics of the envelope protein of CHIKV. In addition the study also provides information on the dynamics of interaction of E2 protein with entry inhibitors that will contribute towards structure based drug design.
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.
Background Hepatitis E is a liver disease caused by the pathogen hepatitis E virus (HEV). The largest polyprotein open reading frame 1 (ORF1) contains a nonstructural Y-domain region (YDR) whose activity in HEV adaptation remains uncharted. The specific role of disordered regions in several nonstructural proteins has been demonstrated to participate in the multiplication and multiple regulatory functions of the viruses. Thus, intrinsic disorder of YDR including its structural and functional annotation was comprehensively studied by exploiting computational methodologies to delineate its role in viral adaptation. Results Based on our findings, it was evident that YDR contains significantly higher levels of ordered regions with less prevalence of disordered residues. Sequence-based analysis of YDR revealed it as a “dual personality” (DP) protein due to the presence of both structured and unstructured (intrinsically disordered) regions. The evolution of YDR was shaped by pressures that lead towards predominance of both disordered and regularly folded amino acids (Ala, Arg, Gly, Ile, Leu, Phe, Pro, Ser, Tyr, Val). Additionally, the predominance of characteristic DP residues (Thr, Arg, Gly, and Pro) further showed the order as well as disorder characteristic possessed by YDR. The intrinsic disorder propensity analysis of YDR revealed it as a moderately disordered protein. All the YDR sequences consisted of molecular recognition features (MoRFs), i.e., intrinsic disorder-based protein–protein interaction (PPI) sites, in addition to several nucleotide-binding sites. Thus, the presence of molecular recognition (PPI, RNA binding, and DNA binding) signifies the YDR’s interaction with specific partners, host membranes leading to further viral infection. The presence of various disordered-based phosphorylation sites further signifies the role of YDR in various biological processes. Furthermore, functional annotation of YDR revealed it as a multifunctional-associated protein, due to its susceptibility in binding to a wide range of ligands and involvement in various catalytic activities. Conclusions As DP are targets for regulation, thus, YDR contributes to cellular signaling processes through PPIs. As YDR is incompletely understood, therefore, our data on disorder-based function could help in better understanding its associated functions. Collectively, our novel data from this comprehensive investigation is the first attempt to delineate YDR role in the regulation and pathogenesis of HEV.
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