Orientation Determination of Membrane-Disruptive Proteins Using Powder Samples and Rotational Diffusion: A Simple Solid-State NMR Approach

Abstract

The orientation of membrane proteins undergoing fast uniaxial rotation around the bilayer normal can be determined without macroscopic alignment. We show that the motionally averaged powder spectra exhibit their 0° frequency, [Formula: see text], at the same position as the peak of an aligned sample with the alignment axis parallel to the magnetic field. This equivalence is exploited to determine the orientation of a β-sheet antimicrobial peptide not amenable to macroscopic alignment, using (13)CO and (15)N chemical shifts from powder spectra. This powder sample approach permits orientation determination of naturally membrane-disruptive proteins in diverse environments and under magic-angle spinning.

Figure 1

Schematics showing the equivalence between the 0°-aligned spectra and unoriented spectra. (a) Rigid 0°-aligned sample. (b, c) Mobile aligned sample. In (c), the motionally averaged tensor has the unique axis along the bilayer normal. (d) Mobile unoriented sample.

Figure 2

Calculated ¹³CO (a-d) and ¹⁵N (e-h) powder spectra (solid lines) and 0°-aligned spectra (dotted lines) of a uniaxially mobile β-sheet peptide for various orientations. (a, e) τ = 90°, ρ = 0°. (b, f) τ = 90°, ρ = 90°. (c, g) τ = 45°, ρ = 0°. (d, h) τ = 0°, ρ = 0°. Note the identity between the frequency of the 0°-aligned spectra and the ${\overline{\delta }}_{//}$ position of the powder spectra.

Figure 3

¹³CO spectra of Val₁₆-labeled PG-1 in DLPC membrane. (a) 0°-aligned spectrum from ref. (7). (b) Powder spectrum obtained with CP. (c) Difference spectrum after subtracting the lipid background signal, showing only the peptide signal. (d) Spectrum of a 90°-aligned sample from ref. (7). The peptide signals in (a) and (d) are indicated by an asterisk.

Figure 4

¹⁵N (a, b) and ¹³CO (d, e) static powder spectra of ¹⁵N-Phe₄ and ¹³CO-Val₆ labeled TP-I in DLPC membrane (1:15 molar ratio). ¹⁵N spectrum at 303 K (a) and 243 K (b) differ in the CSA. (c) Simulated rigid-limit ¹⁵N powder pattern. (d) ¹³CO spectrum of the peptide and the lipids at 303 K. (e) ¹³CO spectrum of the peptide after subtracting the lipid background signal. (f) Simulated rigid-limit ¹³CO spectrum.

Figure 5

TP-I orientation from ¹⁵N and ¹³CO chemical shifts. (a) Phe₄ ¹⁵N chemical shift as a function of (θ, φ) of the bilayer normal in the molecule-fixed PDB system. (b) Val₆ ¹³CO chemical shift as a function of (θ, φ). The measured chemical shifts with the associated uncertainty are colored. (c) The ¹³CO and ¹⁵N chemical shifts overlap at (θ, φ) = (78°, 155°). (d) TP-I orientation with the bilayer normal in the vertical direction. The peptide and DLPC bilayers are drawn to scale.