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 (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.

Figure 1

(A) Schematic showing the amino acid sequences of the channel-forming transmembrane domain (M2 domain, δ subunit) of the TOAC1 AChR and TOAC18 AChR peptide, where τ is the TOAC spin label. (B) Schematic showing TOAC18 AChR as a linear α-helical peptide with the principal axes of the TOAC spin label moiety being labeled as follows: the z-axis is directed along the p–orbital, the x-axis is directed along the N-O bond and the y-axis is perpendicular to the other axes. (C) The peptide is shown in the transmembrane orientation, where h is nearly collinear (helical tilt of 12°) with the normal to the phospholipids bilayers n based on ¹⁵N solid-state NMR studies.

Figure 2

EPR spectra of (A) TOAC1 AChR and (B) TOAC18 AChR in randomly dispersed DMPC/DHPC bicelles. Spectra show the dramatic differences in the nanosecond rotational dynamics, from the narrow linewidth spectrum of TOAC1 (dynamic disorder, 1.2 × 10⁻⁹ s; (A)) to the broad spectrum of the spin label at position 18 (B), indicating highly restricted motion (1.3 ×10⁻⁸ s).

Figure 3

Spin-labeled X-band EPR spectra of TOAC18 AChR incorporated into either magnetically aligned (A and B) or randomly dispersed (C) phospholipid bilayers. For magnetically aligned samples, the average normal to the phospholipid bilayers, n, were either parallel (A) or perpendicular (B) to the applied magnetic field, B₀.

Figure 4

Side view scheme (A) and top view structure (B) of AChR-M2δ in the parallel-aligned bilayers show the relationship between the director tilt angle (ζ) as an angle between n and the director axis Z_D, helical tilt angle (φ) as an angle between n and the helical axis h, and the angle of 21° between h and Z_D. The top view figure (B) shows that the plane (n, h) is nearly orthogonal to the (h, Z_D) plane.