Analysis of Conformation and Dynamics of Membrane-Associated Myristoylated HIV-1 Nef

The accessory protein HIV-1 Nef is crucial for the progression of AIDS following HIV infection. Anchoring of HIV-1 Nef to the inner leaflet of the cell membrane is essential for many of Nef major functions, including cell signaling disruption and internalizing cell surface receptors. Membrane anchoring is accomplished via N-terminal myristoylation, a covalent acylation modification and electrostatic attraction of select N-terminal residues. Disruption of this modification has been shown to dramatically impair Nef's functions and reduce HIV infectivity.;The shape profile of membrane bound on full-length myristoylated Nef was determined using Neutron Reflectometry (NR) and Langmuir monolayers combined with molecular modeling. Isotopically labeled myristoylated Nef was expressed and purified to increase the fidelity of the final neutron reflectometry model. The overall membrane bound profile of Nef was probed at various lipid packing densities, including 20 to 35 mN/m. It was found that at high (35 mN/m) lipid packing densities, the myristate moiety was unable to insert into the lipid membrane and Nef adopted what was believed to be a more "closed conformation" where the majority of the protein was suspended roughly 50 A from the lipid head groups. Conversely, at low (20 mN/m) lipid packing densities Nef adopted a more "open conformation". In this state, the myristate and hydrophobic residues on the N-terminal arm was able to insert into the membrane and the core of Nef was suspended roughly 100 A from the lipid headgroups.;In order to better refine details of membrane associated Nef, a new method combining the Langmuir monolayer system (employed in NR measurements) and hydrogen deuterium exchange mass spectrometry (HDX MS) was developed. Method development and validation were completed to assess the utility and applicability as an analytical technique. In order to confirm that this new method could be used for HDX, the deuterium recovery had to be monitored. There was no significant loss of deuterium label using the Langmuir monolayer HDX MS method when compared to a more conventional, solution based format. The method was further validated by observing known lipid-induced structural changes in the peptide melittin and the myristoyl-switch protein Arf-1. In both experiments, the reproducibility and quality of the data remained high and were equivalent with conventional solution HDX MS methods.;After validation of this HDX MS method involving Langmuir monolayers, the conformation of full-length myristoylated Nef at the membrane was investigated. At low lipid packing densities, Nef peptides in the N-terminal arm and the C-terminal loops showed significant protection upon membrane association, whereas the core was exposed. The deuterium incorporation of Nef at a high and low lipid packing density was compared and showed that Nef was significantly more exposed to labeling in the low lipid packing environment. Both observations are consistent with the previously determined neutron reflection models with regards to the observed "open" and "closed" conformations. The new HDX MS method can be applied to other peripheral membrane proteins to resolve their membrane bound dynamics. This novel method can also be combined with other orthogonal techniques to further expand the conformational details of membrane-bound proteins.