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.

Recombinant proteins (RPs) are commonly expressed in bacteria followed by solubilization and chromatography. Purified RP yield can be diminished by losses at any step with very different changes in methods needed to try to improve yield. Time and labor can therefore be saved by first identifying the specific reason for low yield. The present study describes a new solid-state NMR approach to RP quantitation in whole cells or cell pellets without solubilization or purification. The approach is straightforward and inexpensive and only requires ~50 mL culture and a low-field spectrometer.

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