Determining the Topology of Integral Membrane Peptides Using EPR Spectroscopy

Abstract

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 (M2δ) 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° was calculated for the M2δ peptide with respect to the bilayer normal of the membrane, which agrees well with previous 15 N 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 15 N solid-state NMR spectroscopy; thus, the helical tilt of an integral membrane peptide can be determined with only 100 μg of peptide. The helical tilt can be determined more accurately by placing TOAC spin labels at several positions with this technique.

Probing topology and dynamics of the second transmembrane domain (M2δ) of the acetyl choline receptor using magnetically aligned lipid bilayers (bicelles) and EPR spectroscopy

“…As indicated in the previous work, data from several peptide substitutions provide a more accurate value of helical tilt for a peptide. 12 From the data presented in this paper, the helical tilt calculated to be 9–17° falls within ±4° of the established value. 13,14 Additionally, by using multiple peptides an assessment can be made about the secondary structure of the peptide and speed of rotation about the long axis of the integral peptide.…”

Biophysical EPR Studies Applied to Membrane Proteins

“…Nitroxide based CW-EPR spectroscopy at X-band can also be used to study membrane topology of membrane proteins/peptides bound to aligned phospholipid bilayers [51,52]. Recently, the Lorigan lab used the membrane alignment technique coupled with dipolar broadening CW-EPR to determine the distance and relative orientation of two nitroxide spin labels on the integral membrane peptide (M2δ segment of the acetylcholine receptor (AchR)) and peripheral membrane peptide (antimicrobial peptide magainin-2) in 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC) vesicles [52,53].…”

Optimization of bicelle lipid composition and temperature for EPR spectroscopy of aligned membranes

“…In a well-prepared sample containing many bicelles, the alignment homogeneity is excellent, providing a more physiological alternative to other anisotropic membrane systems such as partially dehydrated lipid bilayers supported on glass plates [4] or lipid-containing crystals [5]. Since transmembrane proteins can be uniformly oriented in an aligned bicelle sample, techniques with sensitivity to orientation, such as nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR), can be used to measure probe orientation with high accuracy relative to the applied magnetic field [6-8], thus providing crucial information about protein structure and dynamics relative to the membrane.…”