Revealing the Structural and Molecular Basis of Retroviral Assembly and Endolysin PlyC Membrane Translocation Using Surface Plasmon Resonance and Neutron Reflectometry

In this thesis we focus on the mechanistic understanding of two membrane mediated biological processes: retroviral assembly and endolysin PlyC plasma membrane translocation. Retroviral Gag polyprotein is the structural determinant that assembles in a protein lattice on the hosts plasma membrane to trigger formation of the viral protein/membrane shell. In this process, it employs multiple signals electrostatic, hydrophobic and lipid-specific interactions conferred by the matrix domain (MA) to recruit the protein to the proper cellular location and assist proteinprotein interactions located on full-length Gag in lattice formation. In this thesis I address the interaction of non-lipidated and myristoylated (+myr) HIV-1 Gag matrix domains with model membranes, sparsely tethered bilayer membranes (tBLMs), to dissect these complex membrane signals and quantify their contributions to the overall interaction. SPR on well-defined membrane models is used to quantify binding affinities and amounts of protein and yields free binding energy contributions, ∆G, of the various signals. Charge-charge interactions in the absence of the phosphatidylinositide PI(4,5)P2 attract the protein to acidic membrane surfaces, and myristoylation increases the affinity by a factor of 10, arguing against a PI(4,5)P2-trigger of myristate exposure. Lipid-specific interactions with PI(4,5)P2, the major signal lipid in the inner plasma membrane, increase membrane attraction at a similar level as the protein lipidation. Cholesterol augments protein affinity strongly, apparently by enabling efficient myristate insertion and PI(4,5)P2 binding. We thus observe that the isolated matrix protein, in the absence of proteinprotein interaction conferred by the full-length Gag, binds the membrane with sub-micromolar affinities. These studies resulted in a detailed understanding of the thermodynamic aspects that determine membrane association, preferential lipid recruitment to the viral shell, and aspects of Gag assembly into the membrane-bound protein lattice that are driven by its matrix domain We also report the difficulties in the assessment of protein-membrane complex structures that involved HIV-1 +myrMA. While myrMA is well behaved, and protein-membrane complexes could be readily characterized by neutron reflection (NR) on stBLMs , this was not possible for +myrMA because of membrane reconstruction that prevented the interpretation of NR data after protein incubation of the bilayer. We confirm the hypothesis of membrane reconstruction with electron microscopy and screen for experimental conditions that would allow the maintainance of planar bilayers needed for NR after +myrMA incubation. We are able to identify a pH range near pH = 8 where membrane reconstruction is minimal. The mechanisms by which full-length Gag proteins interact with membranes were also investigated. Rous sarcoma virus (RSV) Gag protein, and Murine leukemia virus (MLV) had their biophysical properties and membrane interactions in tBLMs studied by SPR and NR. We correlated solution and membrane-bound structures of the Gag proteins from various retroviral species and compare to mutants lacking the linker that connects MA and CA, MLV ∆P12 and RSV ∆flex. MLV wild-type and ∆P12 mutant shows similar affinities to anionic membranes. However, structural characterization by NR found significant differences, ∆P12 extended 200 Å away from the bilayer surface while the wild-type extended only 100 Å. RSV Gag was found to have 10 and 100 fold higher affinity for charged membranes compared to wild-type MLV and RSV MA, respectively. We also demonstrate that RSV wild-type and ∆flex mutant have similar affinity and conformation at anionic membranes with the interaction between RSV Gag and membranes being strongly cooperative. The NR studies indicate that RSV Gag in the membrane-