Determining the topology of integral membrane peptides using EPR spectroscopy.
This paper reports on the development of a new structural biology technique for determining the membrane topology of an integral membrane protein inserted into magnetically aligned phospholipid bilayers (bicelles) using EPR spectroscopy. The nitroxide spin probe, 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC), was attached to the pore-lining transmembrane domain (M2delta) of the nicotinic acetylcholine receptor (AChR) and incorporated into a bicelle. The corresponding EPR spectra revealed hyperfine splittings that were highly dependent on the macroscopic orientation of the bicelles with respect to the static magnetic field. The helical tilt of the peptide can be easily calculated using the hyperfine splittings gleaned from the orientational dependent EPR spectra. A helical tilt of 14 degrees was calculated for the M2delta peptide with respect to the bilayer normal of the membrane, which agrees well with previous 15N solid-state NMR studies. The helical tilt of the peptide was verified by simulating the corresponding EPR spectra using the standardized MOMD approach. This new method is advantageous because: (1) bicelle samples are easy to prepare, (2) the helical tilt can be directly calculated from the orientational-dependent hyperfine splitting in the EPR spectra, and (3) EPR spectroscopy is approximately 1000-fold more sensitive than 15N solid-state NMR spectroscopy; thus, the helical tilt of an integral membrane peptide can be determined with only 100 microg of peptide. The helical tilt can be determined more accurately by placing TOAC spin labels at several positions with this technique.
Inbaraj
Johnson J
JJ
Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, USA.
Cardon
Thomas B
TB
Laryukhin
Mikhail
M
Grosser
Stuart M
SM
Lorigan
Gary A
GA
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000737:chemistry*; D003198:Computer Simulation; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D011487:Protein Conformation; D011978:Receptors, Nicotinic / Q000737:chemistry*; D013113:Spin Labels
D011978:Receptors, Nicotinic; D013113:Spin Labels