Influenza virus causes substantial public health risk worldwide. Influenza is an enveloped virus and hemagglutinin membrane protein present in the viral membrane plays an important role in the viral infection process. Hemagglutinin protein is composed of two subunits called HA1 and HA2. Binding of the virus to the host cell is governed by HA1 subunit, and HA2 subunit is responsible for the fusion of viral membrane and endosomal membrane.My research has focused on production and characterization of several protein constructs containing different domains (with or without FP or with or without TM) and the characterization of the full length HA2. Biophysical comparisons between full-length HA2 and shorter constructs including SHA2, FHA2, and SHA2-TM were performed. Biophysical characterization techniques such as CD spectroscopy, SEC, crosslinking experiments, mass spectrometry and vesicle fusion assays were used in this study. Physiologically relevant oligomeric states of these hemagglutinin constructs were identified. At pH 7.4, the physiological pH, these constructs are trimeric helical molecules in detergents. The melting temperature for full length HA2 is > 90 oC in decylmaltoside (DM) representing a highly thermostable structure. All constructs are positively charged at pH 5.0 and induce vesicle fusion with negatively-charged vesicles. However, fusion with negatively charged vesicles at pH 7.4 was negligible. With positively charged vesicles, pH-dependence was reversed leading to conclude that attractive protein/vesicle electrostatics play a role in fusion between vesicles and hemagglutinin constructs.My work will help future scientists set up crystallography experiments since this is the first time that full length HA2 has been produced in mg quantities using bacterial expression system. Furthermore, it is hypothesized that in the final stage of the fusion of full length HA2 constructs as well as full length HIV gp41 constructs there is a formation of complex in between FP and TM. But, still there is no experimental evidence for support this hypothesis. HDX experiment completed and proposed in this dissertation will help to investigate this hypothesis.
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