Control of Transmembrane Helix Dynamics by Interfacial Tryptophan Residues

Abstract

Transmembrane protein domains often contain interfacial aromatic residues, which may play a role in the insertion and stability of membrane helices. Residues such as Trp or Tyr, therefore, are often found situated at the lipid-water interface. We have examined the extent to which the precise radial locations of interfacial Trp residues may influence peptide helix orientation and dynamics. To address these questions, we have modified the GW^5,19ALP23 (acetyl-GGALW⁵(LA)₆LW¹⁹LAGA-[ethanol]amide) model peptide framework to relocate the Trp residues. Peptide orientation and dynamics were analyzed by means of solid-state nuclear magnetic resonance (NMR) spectroscopy to monitor specific ²H- and ¹⁵N-labeled residues. GW^5,19ALP23 adopts a defined, tilted orientation within lipid bilayer membranes with minimal evidence of motional averaging of NMR observables, such as ²H quadrupolar or ¹⁵N-¹H dipolar splittings. Here, we examine how peptide dynamics are impacted by relocating the interfacial Trp (W) residues on both ends and opposing faces of the helix, for example by a 100° rotation on the helical wheel for positions 4 and 20. In contrast to GW^5,19ALP23, the modified GW^4,20ALP23 helix experiences more extensive motional averaging of the NMR observables in several lipid bilayers of different thickness. Individual and combined Gaussian analyses of the ²H and ¹⁵N NMR signals confirm that the extent of dynamic averaging, particularly rotational "slippage" about the helix axis, is strongly coupled to the radial distribution of the interfacial Trp residues as well as the bilayer thickness. Additional ²H labels on alanines A3 and A21 reveal partial fraying of the helix ends. Even within the context of partial unwinding, the locations of particular Trp residues around the helix axis are prominent factors for determining transmembrane helix orientation and dynamics within the lipid membrane environment.

Figure 1

Molecular models of GWALP-like peptides. From left to right, W^2,3,21,22ALP23, W^2,22W^5,19ALP23, GW^5,19ALP23, and GW^4,20ALP23 are shown. For amino acid sequences, see Table 1. The model for GW^4,20ALP23 was rotated 90°. To see this figure in color, go online.

Figure 2

Circular dichroism spectra for GW^4,20ALP23 in DLPC (red), DMPC (blue), and DOPC (black) vesicles. To see this figure in color, go online.

Figure 3

31-Phosphorous NMR spectra for GW^4,20ALP23 in (A) oriented DMPC bilayers and (B) DMPC/DHPC bicelles. To see this figure in color, go online.

Figure 4

Separated local-field PISEMA spectra. (A) ¹⁵N/¹H-separated local-field spectrum for GW^4,20ALP23 is shown. The sample is oriented in DMPC/DHPC bicelles and contains ¹⁵N-labeled residues 13–17 (assigned as depicted). (B) A one-dimensional ¹⁵N NMR spectrum for GW^4,20ALP23 is shown.

Figure 5

Deuterium quadrupolar splittings for GW^4,20ALP23 in oriented DLPC, DMPC, and DOPC bilayers for samples oriented with β = 90°. The ²H-labeled alanine identities are, from top to bottom, (5,7), (9,11), (13,15), (17,19), and (3,21), with the first alanine of each pair 100% deuterated and the second alanine 50% deuterated.

Figure 6

Helix dynamics illustrated by separated local-field PISEMA spectra. (A) Data and PISA wheel for GW^5,19ALP23 are shown. (B) The PISA wheel from (A) is repeated along with data showing extensive motional averaging for W^2,22W^5,19ALP23 (blue contours) and for GW^4,20ALP23 (black contours). Each sample is oriented in DMPC/DHoPC bicelles with β = 90° and is ¹⁵N-labeled in residues 13–17 (assigned as indicated). The spectra for GW^5,19ALP23 and W^2,22W^5,19ALP23 were recorded previously (17, 18). To see this figure in color, go online.

Figure 7

“Apparent” GALA quadrupolar wave plots for transmembrane peptide helices in DLPC (red circles), DMPC (blue triangles), or DOPC (black squares) oriented bilayer membranes. (A) GW^5,19ALP23, (B) Y^4,5GW¹⁹ALP23, and (C) GW^4,20ALP23 are shown. (C) denotes the positions of the deuterium-labeled alanine residues. Only (A) reflects correctly the variation of the helix tilt τ₀ and the constant helix azimuthal rotation ρ₀ in the different bilayers. The amplitudes and phases of the waves in (B) and (C) reflect excessive dynamic averaging of the ²H quadrupoles. See text for details. See also Table 4. To see this figure in color, go online.

Figure 8

Model to describe transmembrane peptide motions and dynamics with respect to the bilayer normal B₀. The apparent average helix tilt and rotation are denoted by τ₀ and ρ₀, respectively, and the oscillations about these orientations are shown as the helical “wobble” (σ_τ) and rotational “slippage” (σ_ρ). To see this figure in color, go online.

Figure 9

Comparison of the “apparent” GALA quadrupolar wave plots for GW^4,20ALP23 in DMPC oriented bilayers (black circles) and DMPC/DHPC bicelles (red triangles). Positions 3 and 21 were left out of both analyses. To see this figure in color, go online.