Conformational mapping of the N-terminal peptide of HIV-1 gp41 in membrane environments using (13)C-enhanced Fourier transform infrared spectroscopy.
The N-terminal domain of HIV-1 glycoprotein 41000 (FP; residues 1--23; AVGIGALFLGFLGAAGSTMGARSCONH(2)) participates in fusion processes underlying virus--cell infection. Here, we use physical techniques to study the secondary conformation of synthetic FP in aqueous, structure-promoting, lipid and biomembrane environments. Circular dichroism and conventional, (12)C-Fourier transform infrared (FTIR) spectroscopy indicated the following alpha-helical levels for FP in 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) liposomes-hexafluoroisopropanol (HFIP)>trifluoroethanol (TFE)>phosphate-buffered saline (PBS). (12)C-FTIR spectra also showed disordered FP structures in these environments, along with substantial beta-structures for FP in TFE or PBS. In further experiments designed to map secondary conformations to specific residues, isotope-enhanced FTIR spectroscopy was performed using a suite of FP peptides labeled with (13)C-carbonyl at multiple sites. Combining these (13)C-enhanced FTIR results with molecular simulations indicated the following model for FP in HFIP: alpha-helix (residues 3-16) and random and beta-structures (residues 1-2 and residues 17-23). Additional (13)C-FTIR analysis indicated a similar conformation for FP in POPG at low peptide loading, except that the alpha-helix extends over residues 1-16. At low peptide loading in either human erythrocyte ghosts or lipid extracts from ghosts, (13)C-FTIR spectroscopy showed alpha-helical conformations for the central core of FP (residues 5-15); on the other hand, at high peptide loading in ghosts or lipid extracts, the central core of FP assumed an antiparallel beta-structure. FP at low loading in ghosts probably inserts deeply as an alpha-helix into the hydrophobic membrane bilayer, while at higher loading FP primarily associates with ghosts as an aqueous-accessible, beta-sheet. In future studies, (13)C-FTIR spectroscopy may yield residue-specific conformations for other membrane-bound proteins or peptides, which have been difficult to analyze with more standard methodologies.
Gordon
Larry M
LM
Department of Pediatrics, Harbor-University of California at Los Angeles Medical Center, 90502-2064, USA. lgordon2@san.rr.com
Mobley
Patrick W
PW
Pilpa
Rosemarie
R
Sherman
Mark A
MA
Waring
Alan J
AJ
D000595:Amino Acid Sequence; D002021:Buffers; D002247:Carbon Isotopes; D002942:Circular Dichroism; D004910:Erythrocyte Membrane / Q000737:chemistry*; D015700:HIV Envelope Protein gp41 / Q000737:chemistry*; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry*; D008563:Membrane Lipids / Q000737:chemistry; D008969:Molecular Sequence Data; D010715:Phosphatidylglycerols; D020005:Propanols; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared; D014270:Trifluoroethanol; D006845:Hydrocarbons, Fluorinated
D002021:Buffers; D002247:Carbon Isotopes; D015700:HIV Envelope Protein gp41; D008051:Lipid Bilayers; D008563:Membrane Lipids; D010715:Phosphatidylglycerols; D020005:Propanols; D014270:Trifluoroethanol; C001337:hexafluoroisopropanol; D006845:Hydrocarbons, Fluorinated