Solid-State Nuclear Magnetic Resonance 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

摘要

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 N-terminal 175 residues are the ectodomain which lies outside the virus. This paper describes production and characterization of an ectodomain construct containing the 154 N-terminal gp41 residues including 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 which have been less studied than European/North American clade B strains. Fgp41 expression at ~100 mg/L 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 culture. SSNMR spectra of membrane-associated Fgp41 showed high helicity at residues C-terminal of the FP. This was consistent with “six-helix bundle” (SHB) structure which is the final gp41 state during membrane fusion. This observation plus 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 evidenced 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.