Membrane fusion is a dynamic and multistep process in which proteins and membranes spend relatively little time at any stage. By using experimental conditions that allow the process to proceed partway, but not all the way to fusion, we capture intermediate states of fusion. Characterizing them both biochemically and biophysically, we infer the conformation of the fusion proteins and the configuration of the lipids at distinct points in the progression toward fusion.
Many fusion proteins form six-helix bundles - a central triple-stranded coiled-coil surrounded by helices that pack into each of the three grooves created by the central core. The bundle structure is critical to fusion, but how? We are investigating this by using the fusion proteins from influenza virus, hemagglutinin, and from HIV, Env. With site directed mutagenesis, peptides that inhibit formation of the bundle, and control of lipids, the point in the fusion process that the bundle forms is being determined. The general themes and the variations within the themes are formulated by using two different proteins, each exhibiting six-helix bundle, but with otherwise very different structures.
Some viruses, such as encephalitis and West Nile, have fusion proteins that do not contain bundles and thus rather different mechanisms of fusion may be expected. We use Semliki Forest virus as a prototypic virus for these classes and have found that its fusion is in several ways, quite different from that of influenza and HIV. Simliki Forest virus requires cholesterol and sphinglolipids in the target membrane for fusion to occur. By fusing cells that express the fusion protein to planar phospholipid bilayer membranes, the lipid requirements are determined conveniently with a system that allow for sensitive electrical measures of the fusion pores.
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