Research

• membrane protein oligomerization and mobility
• inhibitors and antibodies as conformational probes
• binding and fusion

The enveloped viruses including HIV gain access to human cells using an envelope protein complex located on the surface of the virion. In the case of HIV, the transmembrane portion of this protein complex, gp41, is the fusion protein. The globular envelope subunit, gp120, is non-covalently associated to the transmembrane portion and makes initial contact with the cellular receptors. There is evidence that the envelope protein subunits form a trimer of heterodimers that resembles a stalk structure on the virus surface and on the cell surface of infected cells. The structure of the portion of gp41 that protrudes from the viral surface has been solved in vitro. It is a trimer that forms a very stable six-helix bundle. This striking stability has led researchers to posit that the structures solved in vitro are representative of the post-fusion form of gp41 with the energy from the conformational change driving fusion. There are numerous peptide inhibitors and conformational antibodies that bind to gp41 only at different stages during the fusion process. This also suggests that there are distinctly different conformations of gp41 that occur as fusion proceeds. It is my hypothesis that gp41, during formation of a fusion pore, undergoes conformational changes that are dramatic and involve both local structural changes and also changes in oligomerization state. If this is the case, it would be similar to the aggregate of at least eight hemagluttinin molecules that has been found to develop during influenza virus fusion. I use peptide and small molecule inhibitors and conformational antibodies as structural probes to dissect the sequence of changes that occur to the complex during fusion. This work combines the classical biochemistry techniques of protein cross-linking, glycerol gradient ultracentrifugation with the more recent developments of infrared fluorescence imaging and in cell westerns. The details of the conformational changes that occur in the envelope complex during fusion are vital information from which researchers can design different and improved inhibitors that will be beneficial on both a therapeutic and a prophylactic level. It is my objective in my career to provide vital basic research in virology and cell biology in order to merge in vitro structural studies with increasingly greater resolution of protein machines in their native cellular environment.

• membrane fusion protein reconstitution
• targeting cancer cells
• targeting viruses

Liposomes have multifunctional capacity which makes them very promising tools in the treatment of cancers, HIV/AIDS, and in DNA delivery for genetic disorders. One of the advantages of liposomes is that they can compartmentalize hydrophobic components in their lipid bilayers along with being able to encapsulate hydrophilic components. This gives researchers the possibility to incorporate different modalities into one formulation including molecules for targeting, biomarker detection, in vivo imaging and delivery of chemotherapy agents. One of the major drawbacks in using liposomes as drug carriers, however, is that the entry mechanism and the subsequent fate of liposomes after entry into cells is not known from the outset. Many liposomal formulations act simply as transfection reagents by binding molecules of positive charge, such as DNA, to their phospholipid head groups. The DNA is the transferred by an indeterminate merging of the lipid and the DNA molecules. Many of the other types of liposomal formulations are taken up by endocytosis and shuttled unpredictably to intracellular vesicles which might have different chemical compositions and therefore lead to unpredictable delivery. I am overcoming this drawback by developing innovative nanoparticles that deliver therapeutic agents directly into the cytosol of targeted cells utilizing direct membrane fusion with the plasma membrane. I am also targeting these systems to specific cell types by the addition of targeting moieties. It is projected that via this route of protein and nanoparticle engineering, it will eventually be possible to target not only specific cells but to target specific organelles within cells such as the nucleus, Golgi apparatus or endoplasmic reticulum. This development will provide prospective treatments for diverse diseases from viruses to cancer and will also lead to the advancement of basic research in membrane proteins and membrane cellular biology.