title: 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.
abstract: 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 (13)CO and (15)N 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.
authors: Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA.
authors: Erica P
authors: EP
authors: Vogel
authors: Jaime
authors: J
authors: Curtis-Fisk
authors: Kaitlin M
authors: KM
authors: Young
authors: David P
authors: DP
authors: Weliky
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D005612:Freeze Drying; D015870:Gene Expression; D006678:HIV / Q000737:chemistry* / Q000235:genetics; D015700:HIV Envelope Protein gp41 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1; D006801:Humans; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
chemical_list: D015700:HIV Envelope Protein gp41; D011993:Recombinant Fusion Proteins