American Chemical Society
Browse
bi201292e_si_001.pdf (305.17 kB)

Solid-State Nuclear Magnetic Resonance (NMR) Spectroscopy of Human Immunodeficiency Virus gp41 Protein That Includes the Fusion Peptide: NMR Detection of Recombinant Fgp41 in Inclusion Bodies in Whole Bacterial Cells and Structural Characterization of Purified and Membrane-Associated Fgp41

Download (305.17 kB)
journal contribution
posted on 2016-02-22, 13:51 authored by Erica P. Vogel, Jaime Curtis-Fisk, Kaitlin M. Young, David P. Weliky
Human immunodeficiency virus (HIV) infection of a host cell begins with fusion of the HIV and host cell membranes and is mediated by the gp41 protein, a single-pass integral membrane protein of HIV. The 175 N-terminal residues make up the ectodomain that lies outside the virus. This work describes the production and characterization of an ectodomain construct containing the 154 N-terminal gp41 residues, including the fusion peptide (FP) that binds to target cell membranes. The Fgp41 sequence was derived from one of the African clade A strains of HIV-1 that have been less studied than European/North American clade B strains. Fgp41 expression at a level of ∼100 mg/L of culture was evidenced by an approach that included amino acid type 13CO and 15N labeling of recombinant protein and solid-state NMR (SSNMR) spectroscopy of lyophilized whole cells. The approach did not require any protein solubilization or purification and may be a general approach for detection of recombinant protein. The purified Fgp41 yield was ∼5 mg/L of culture. SSNMR spectra of membrane-associated Fgp41 showed high helicity for the residues C-terminal of the FP. This was consistent with a “six-helix bundle” (SHB) structure that is the final gp41 state during membrane fusion. This observation and negligible Fgp41-induced vesicle fusion supported a function for SHB gp41 of membrane stabilization and fusion arrest. SSNMR spectra of residues in the membrane-associated FP provided evidence of a mixture of molecular populations with either helical or β-sheet FP conformation. These and earlier SSNMR data strongly support the existence of these populations in the SHB state of membrane-associated gp41.

History