John R. Engen
Paul Vouros, Max Diem (1947-), Robert N. Hanson, Mark C. Williams
Date of Award
Doctor of Philosophy
Department or Academic Unit
College of Science. Department of Chemistry and Chemical Biology.
chemistry, HIV/AIDS, Nef, AIDS, HIV, gamma-glutamyl carboxylase (GGCX), myristoylation, association
HIV (Viruses), Membrane proteins, Nanomedicine
Amino Acids, Peptides, and Proteins | Biochemistry | Nanoscience and Nanotechnology
The HIV-1 accessory protein Nef plays a critical role in HIV/AIDS progression. In order for proper function of Nef, it must be localized to the inner leaflet of the cellular membrane. Nef is N-terminally myristoylated, which acts as a membrane anchor. Without this modification, Nef function is crippled as it cannot localize to the membrane. Individuals infected with strains of HIV lacking a functional Nef protein do not progress to AIDS for typically 15-25 years. These individuals are called long-term non-progressors.
The effect of N-terminal myristoylation on Nef was probed by hydrogen exchange mass spectrometry. No differences in hydrogen exchange were detected due to conformational or dynamic changes as a result of the N-terminal modification. This directly contradicts a proposed hypothesis where myristoylated Nef adopts a closed conformation while in solution, with the myristate binding to the core domain and protecting the N-terminal arm.
Nef forms a stable complex with the enzyme which catalyzes its N-terminal myristoylation, human N-myristoyl transferase-1 (NMT). The nature of the interaction between these two proteins was investigated by hydrogen exchange mass spectrometry. While NMT had no effect on Nef, there were significant increases in the deuterium uptake in NMT while bound to Nef. The changes were concentrated around the active site and were determined to be caused by the loss of interactions with myristoyl coenzyme A, not directly due to interactions with Nef. Taken together, this suggests that either the myristoylated Nef-NMT complex is maintained by some non-HX altered force or by the myristate of Nef remaining in the active site of NMT as no protein:protein interaction surface could be detected by HX MS.
The structure of N-terminally histidine tagged Nef bound to a lipid monolayer was determined at 7-10 Å by neutron reflectometry. Nef was produced as stable isotope labeled (heavily deuterated at aliphatic positions) in order to increase confidence in the final model. It was determined, with this model system, that the core domain of Nef interacts directly with the membrane and some part of Nef inserts into the lipid layer. These data represent the first structural determination of membrane associated Nef.
In order to apply hydrogen exchange mass spectrometry to membrane associated, myristoylated Nef the nanodisc - HX MS method was developed and validated with gamma-glutamyl carboxylase (GGCX) embedded in nanodiscs. GGCX was chosen because it is a very large protein (770 residues, ~94 kDa) with 5-transmembrane helices and it is much more complex than Nef (221 residues, ~25 kDa) which is peripherally associated to membranes.
To properly understand the nanodisc as a model membrane system, hydrogen exchange into the membrane scaffold protein (MSP) surrounding the edge of the phospholipid bilayer was monitored and compared to lipid-free MSP. The resulting peptic peptides and their deuterium uptake signatures in properly formed nanodiscs were cataloged. The conformational transition of MSP due to lipid-association resulted in significant protection from exchange at the shortest timepoints. However, MSP in nanodiscs displayed a dynamic nature and acquired as much deuterium as lipid-free MSP with only three small regions showing differences greater than 1 Da at 4 hours of labeling. EX1 kinetic signatures were seen in both lipid-free and lipid-associated.
Christopher R. Morgan
Morgan, Christopher R., "HIV-1 Nef: from myristoylation to association with biological membranes" (2011). Chemistry Dissertations. Paper 28. http://hdl.handle.net/2047/d20001053
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