Membrane fusion, mediated by viral spike glycoproteins, is a key process in the infection cycle of all enveloped human and animal viruses. Even though influenza hemagglutinin (HA) is the structurally best studied of all viral (or cellular) fusion proteins, the molecular mechanisms by which this proteins catalyzes membrane fusion are only poorly understood. Therefore, the overall goal of this research is to help elucidate these mechanisms and, particularly, the structural transformations that take place during influenza HA-mediated membrane fusion. A combined biochemical and biophysical approach will be taken to pursue three specific aims, namely: (1) to develop improved fluorescence and infrared spectroscopic methods for studying the structure and dynamics of membrane components as they relate to the mechanism of viral membrane fusion; (2) to determine the dynamic structure of fusion intermediates using strain X31HA as the model protein; and (3) to investigate the structure, dynamics, and interactions of wild-type and mutant fusion and transmembrane peptides and their effect on membrane structure by vibrational and electron paramagnetic resonance spectroscopy. The general methodology to achieve these goals will be the following: Influenza HA will be functionally reconstituted into planar supported phospholipid bilayers. Surface-sensitive techniques such as total internal reflection fluorescence microscopy (TIRFM) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy will be used to structurally investigate the reconstituted fusion complexes. Membrane structures and lipid bilayer distances will be determined by TIRFM at various stages of membrane fusion. Protein conformational changes, including molecular orientations and secondary structure changes of putative fusion intermediates will be studied by ATR-FTIR spectroscopy in situ as a function of pH, lipid composition, temperature and time. Proteolytic fragmentation of HA and spectroscopic studies of synthetic wild-type and mutant fusion and transmembrane peptide will be used to establish detailed structure-function relationships for several segments of the HA molecule. Taken together, these studies will provide a structural and functional basis for understanding the molecular mechanisms of viral spike glyoprotein-mediated membrane fusion.