Journal
PROTEIN ENGINEERING DESIGN & SELECTION
Volume 28, Issue 4, Pages 107-116Publisher
OXFORD UNIV PRESS
DOI: 10.1093/protein/gzv006
Keywords
GB1-gp41 peptide fusion protein; ligand-based NMR screening; NHR groove; overexpression of hydrophobic protein; reverse hairpin gp41 trimer
Funding
- National Institutes of Health [GM087998, P41 RR-01081, AI097051]
- MRSEC program of NSF [DMR-1120901]
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Four new swapped-domain constructs of the ectodomain of human immunodeficiency virus type 1 glycoprotein-41 (gp41) were prepared. The gp41 ectodomain consists of 50-residue N-heptad repeat (NHR), 36-residue disulfide-bonded loop and 39-residue C-heptad repeat (CHR). It folds into a hairpin structure that forms a trimer along the NHR axis. The swapped-domain proteins feature CHR domains of length 39, 28 or 21 residues preceding a 4-residue loop and a 49- or 50-residue NHR domain. The effect of CHR truncation was to expose increasing lengths of the NHR groove, including the conserved hydrophobic pocket, an important drug target. A novel method for preparing proteins with extended exposed hydrophobic surfaces was demonstrated. Biophysical measurements, including analytical ultracentrifugation and ligand-detected Water-Ligand Observed via Gradient Spectroscopy and H-1-N-15-HSQC NMR experiments, were used to confirm that the proteins formed stable trimers in solution with exposed binding surfaces. These proteins could play an important role as receptors in structure-based drug discovery.
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