The lifecycle of the retrovirus HIV-1 is intimately associated with membranes. The virus particles have a membrane envelope that contains structural proteins responsible for recognition and fusion with susceptible cells, and the final step of viral reproduction involves the budding out of new virus particles from the membrane of infected cells. Vpu is an accessory protein encoded in the HIV-1 genome and found in infected cells but not the virus particles. It has two biological activities. It enhances the degradation of CD4/gp160 complexes, enabling gp160 to be processed to the form the gp41 and gp120 proteins required for the formation of new virus particles, and it facilitates the budding out of new virus particles, perhaps through its ion channel activity. Vpu is a small (81 residues) protein and we have developed expression systems in bacteria and have prepared substantial quantities of selectively and uniformly labeled material for NMR experiments. Vpu's biological activities appear to be associated with different regions of the protein structure. It appears to be a typical membrane protein with one hydrophobic membrane spanning helix and several distinct amphipathic regions that are near the surface of the membrane. The solid-state NMR studies of uniformly 15N labeled Vpu oriented in lipid bilayers on glass plates indicate that the protein is immobilized on the time scale of the NMR orientation dependent interactions. The interpretation of the 15N chemical shift anisotropy (CSA) and 15N-1H dipolar couplings of the backbone amide sites reveals that the protein has two helical regions, one that is in the plane of the bilayer and one that is transmembrane. The goal of our research is to determine the complete three-dimensional structure of both phosphorylated and unphosphorylated Vpu in membrane bilayers by solid-state NMR spectroscopy. These studies should give a great deal of insight into the fundamental principles of the structures of membrane proteins and how they are altered by modifications such as phosphorylation.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Biotechnology Resource Grants (P41)
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University of Pennsylvania
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