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 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.

NMR of Macromolecular Assemblies and Machines at 1 GHz and Beyond: New Transformative Opportunities for Molecular Structural Biology

“…Lippens and co-workers presented a method for combined J- and CP-based correlation with 13 CO detection at 900 MHz for characterization of disordered α-synculein in E. coli cells (102). Whole cell solid-state NMR studies have covered a wide range of systems including bacteria and biofilms to address many significant questions including perturbations in bacterial cell walls induced by drug binding (180, 181), cellular composition (182) and metabolomics (183), as well as monitoring of protein expression and cellular processes (184–186). These studies are essential to address pressing issues such as antibiotic resistance (187–189).…”

Magic angle spinning NMR of viruses

“…MAS NMR probed the secondary structure of the fusion peptide in these large gp41 constructs, and molecules with either α-helical or predominantly antiparallel β-sheet conformation were observed in both [79, 192]. Another study from the Weliky group introduced an approach to examine lyophilized whole cells containing an expressed 154-residue gp41 fragment using MAS NMR, and detected helical structure at the C-terminal of this protein construct by REDOR experiments [193]. In addition to the studies on fusion peptides, MAS NMR was also applied to investigate the V3 loop in gp120 and its interaction with antibody fragments [194, 195].…”

Nuclear Magnetic Resonance as a Tool to Characterize the Interactome of Heme Proteins

“…Preliminary experiments on proteins deposited in inclusion bodies in E. coli have demonstrated the potential of in-cell ssNRM. 212,213 Furthermore, the in-cell ssNMR structural characterization of the LR11 (sorLA) transmembrane domain in native E. coli membranes has been performed. 214 In 2012 Glaubitz et al 215 presented an in-cell ssNMR approach to study macromolecules that tumble too slowly for the solution NMR detection, using E. coli thioredoxin and human FK506-binding protein as model systems.…”