Membrane fusion plays a crucial role in many biological processes from virus infection to release of neurotransmitters (Hughson 1999). Membrane -bound surface glycoproteins are involved in the fusion process. The enveloped animal virus infection is initiated by interactions between the virus and the cell membrane through the surface glycoproteins called fusion glycoproteins (Eckert and Kim 2001). The fusion glycoproteins are responsible for both receptor binding and membrane fusion activity. The fusion proteins are characterized by a large ectodomain containing fusion peptides, a transmembrane (TM) domain, and a cytoplasimic domain. The viruses can enter cells either at neutral pH or at acidic pH. When exposed to appropriate conditions, the fusion protein undergoes conformational changes, which in turn drives the fusion process. The fusion glycoproteins can be classified as Class I and Class II fusion proteins (Lescar eta/. 2001 ). The Class I fusion proteins are synthesized as a precursor molecule, which then undergoes proteolytic cleavage to generate a mature molecule containing the hydrophobic fusion peptide at the N -terminal. The class II fusion glycoproteins are not synthesized as precursor molecules, and they have internal fusion peptides. The vesicular stomatitis virus (VSV) glycoprotein G is a class Ill fusion protein. It has a neutral internal fusion peptide and upon exposure to low pH, the protein undergoes reversible conformational change (Gaudin 2000, Yao eta/. 2003). A 62kDa soluble ectodomain of VSV G (Gs) has been generated by limited trypsin digestion. The SDS PAGE gel electrophoresis indicates that the trypsin has possibly cleaved near the transmembrane (TM) domain. Liposome binding experiment suggests that Gs can bind to liposomes in a pH dependent manner. Liposome fusion studied by RET assay suggests that the Gs can induce significant amount of hemifusion. However, it failed to induce any content mixing mainly due to considerable amount of membrane leakage activity. This indicates that the binding to the membrane through the TM domain is required for complete membrane fusion. Unlike TBE E soluble ectodomain, Gs can form dimers and trimers at neutral and fusion active pH. Light scattering experiment shows that the aggregation of Gs increases with a decrease in pH. The conformational change with changes in pH was evident from the trypsin sensitivity assay and CD spectroscopy. It was observed that Gs became resistant to trypsin digestion at low pH and a-helicity content of the molecule increased upon lowering the pH. However, the maximum amount of a-helicity was observed at pH 6. The removal of the TM domain also shifts the optimum fusion pH towards more acidic pH in comparison to VSV G. These results indicate that the TM domain is not required for the oligomerization of G protein, but some role has been reserved for the TM domain during membrane fusion. The CD spectroscopic data also indicated that the G protein undergoes structural rearrangement between pH 7.4-6, which could be responsible for the exposure of fusion peptide and subsequent target membrane binding. / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/22687 |
| Date | 01 1900 |
| Creators | Das, Rahul |
| Contributors | Ghosh, H.P., Biochemistry |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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