Solid-state NMR studies of a diverged microsomal amino-proximate delta12 desaturase peptide reveal causes of stability in bilayer: tyrosine anchoring and arginine snorkeling

Abstract

This study reports the solid-state NMR spectroscopic characterization of the amino-proximate transmembrane domain (TM-A) of a diverged microsomal delta12-desaturase (CREP-1) in a phospholipid bilayer. A series of TM-A peptides were synthesized with 2H-labeled side chains (Ala-53, -56, and -63, Leu-62, Val-50), and their dynamic properties were studied in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) bilayers at various temperatures. At 6 mol % peptide to lipid, 31P NMR spectra indicated that the peptides did not significantly disrupt the phospholipid bilayer in the L(alpha) phase. The 2H NMR spectra from Ala-53 and Ala-56 samples revealed broad Pake patterns with quadrupolar splittings of 16.9 kHz and 13.3 kHz, respectively, indicating restricted motion confined within the hydrocarbon core of the phospholipid bilayer. Conversely, the deuterated Ala-63 sample revealed a peak centered at 0 kHz with a linewidth of 1.9 kHz, indicating increased side-chain motion and solvent exposure relative to the spectra of the other Ala residues. Val-50 and Leu-62 showed Pake patterns, with quadrupolar splittings of 3.5 kHz and 3.7 kHz, respectively, intermediate to Ala-53/Ala-56 and Ala-63. This indicates partial motional averaging and supports a model with the Val and Leu residues embedded inside the lipid bilayer. Solid-state NMR spectroscopy performed on the 2H-labeled Ala-56 TM-A peptide incorporated into magnetically aligned phospholipid bilayers indicated that the peptide is tilted 8 degrees with respect to the membrane normal of the lipid bilayer. Snorkeling and anchoring interactions of Arg-44 and Tyr-60, respectively, with the polar region or polar hydrophobic interface of the lipid bilayer are suggested as control elements for insertional depth and orientation of the helix in the lipid matrix. Thus, this study defines the location of key residues in TM-A with respect to the lipid bilayer, describes the conformation of TM-A in a biomembrane mimic, presents a peptide-bilayer model useful in the consideration of local protein folding in the microsomal desaturases, and presents a model of arginine and tyrosine control of transmembrane protein stability and insertion.

FIGURE 1

Diagram explaining how the helical tilt was determined in a [d₃]Ala peptide. ɛ_‖ is the angle formed by the vector originating from the helical axis that intersects the C_α-C_β alanine bond at the helix backbone, and the helical axis, ɛ_perp, is the angle between the C_α-C_β alanine bond and the vector from the helical axis to the helix backbone as seen in the planar projection of the bond and vector. ρ is the angle of rotation around the helical axis as seen in the planar projection of the vector from the helical axis to the alanine residue to the arbitrary axis in the plane. τ is the tilt angle of the peptide which is shown between the helical axis and the bilayer normal vectors.

FIGURE 2

Amino acid sequence of CREP-1 TM-A, where each amino acid is symbolized by its single-letter code. Peptides were synthesized that contained a single deuterated residue. Labeling locations are shown by the bold, underlined letters.

FIGURE 3

Helical wheel projection of the TM-A CREP1 peptide, with the first amino acid synthesized at the top. The deuterated residues are marked with a box.

FIGURE 4

²H solid-state NMR spectra of a DMPC bilayer incorporated with 6 mol % deuterated TM-A at 45°C. Labeling sites were (A) Val-50, (B) Ala-53, (C) Ala-56, (D) Leu-62, and (E) Ala-63.

FIGURE 5

Simulated spectra of (A) Val-50 and Leu-62, (B) Ala-63, (C) Ala-56 and Ala-53, and (D) low temperature Ala-56 and Ala-53. Simulation parameters are described in the text.

FIGURE 6

²H solid-state NMR spectra of a DMPC/DHPC bicelle samples containing 2 mg TM-A deuterated at Ala-56 acquired at 38°C without (A) and with (B) ytterbium ions. The quadrupolar splitting approximately doubles when comparing the sample with the bilayer normal perpendicular to the magnetic field normal (A) versus the sample with the bilayer normal parallel to the magnetic field normal (B).

FIGURE 7

Model for CREP-1 TM-A in a membrane bilayer based on ²H solid-state NMR spectra of DMPC bilayers infiltrated by TM-A peptides. Residues are labeled assuming a rigid α-helix with 3.6 residues per turn.