Side chain and backbone dynamics of phospholamban in phospholipid bilayers utilizing 2H and 15N solid-state NMR spectroscopy

Abstract

2H and 15N solid-state NMR spectroscopic techniques were used to investigate both the side chain and backbone dynamics of wild-type phospholamban (WT-PLB) and its phosphorylated form (P-PLB) incorporated into 1-palmitoyl-2-oleoyl-sn-glycerophosphocholine (POPC) phospholipid bilayers. 2H NMR spectra of site-specific CD3-labeled WT-PLB (at Leu51, Ala24, and Ala15) in POPC bilayers were similar under frozen conditions (-25 degrees C). However, significant differences in the line shapes of the 2H NMR spectra were observed in the liquid crystalline phase at and above 0 degrees C. The 2H NMR spectra indicate that Leu51, located toward the lower end of the transmembrane (TM) helix, shows restricted side chain motion, implying that it is embedded inside the POPC lipid bilayer. Additionally, the line shape of the 2H NMR spectrum of CD3-Ala24 reveals more side chain dynamics, indicating that this residue (located in the upper end of the TM helix) has additional backbone and internal side chain motions. 2H NMR spectra of both WT-PLB and P-PLB with CD3-Ala15 exhibit strong isotropic spectral line shapes. The dynamic isotropic nature of the 2H peak can be attributed to side chain and backbone motions to residues located in an aqueous environment outside the membrane. Also, the spectra of 15N-labeled amide WT-PLB at Leu51 and Leu42 residues showed only a single powder pattern component indicating that these two 15N-labeled residues located in the TM helix are motionally restricted at 25 degrees C. Conversely, 15N-labeled amide WT-PLB at Ala11 located in the cytoplasmic domain showed both powder and isotropic components at 25 degrees C. Upon phosphorylation, the mobile component contribution increases at Ala11. The 2H and 15N NMR data indicate significant backbone motion for the cytoplasmic domain of WT-PLB when compared to the transmembrane section.

Figure 1

High-resolution solution NMR structures as well as the corresponding sequences for (A) pentameric (bellflower-like assembly) and (B) monomeric (L-shaped) phospholamban, by the Chou (23) and Veglia (26) groups, respectively. The two structures embedded inside membrane bilayers were generated using MOL-MOL and a G5 Apple Mac computer. The three-dimensional structures in panels A and B were obtained from the Protein Data Bank.

Figure 2

Chemical structure of the (A) alanine and (B) leucine amino acids.

Figure 3

²H NMR powder pattern spectra of site-specific CD₃-labeled WT-PLB and its phosphorylated form embedded inside POPC phospholipid bilayers. ²H NMR spectra are shown for (A) PLB-CD₃-Leu51, (B) PLB-CD₃-Ala24, (C) PLB-CD₃-Ala15, and (D) P-PLB-CD₃-Ala15. The experiments were conducted at temperatures ranging from -25 to 60 °C.

Figure 4

²H solid-state NMR spectral simulations using MXQET (55). In the generation of these spectra, rotations along the C_α-C_β bond axis (A), the long peptide helix axis (B and C), and the C_β-C_γ bond axis (D) were considered, and the corresponding differences in ²H NMR spectral line shapes and the quadrupolar splittings (Δν) were compared at different jumps rates. Panel A shows a 3-fold decrease in the quadrupolar splitting (Δν) when faster CD₃-methyl group hopping is considered. In panel A, only the CD₃-methyl group rotation about the C_α-C_β axis was considered. Panel B shows that faster two-site exchange along the peptide helix can gradually change the ²H solid-state NMR spectral line shape from a typical Pake pattern to a bell-shaped pattern. In these simulations, the helix flips between two equally populated symmetry-related rotamers separated by 180°. This type of rotation does not affect the spectral splitting. (C) Simulation corresponding to continuous rotation along the long peptide helix which gradually changes the typical Pake pattern spectrum to an isotropic peak. In these simulations, the helix jumps among three equally populated symmetrical rotomers separated by 120°. Additionally, in panels B and C, the angle between the C_α-C_β bond of the CD₃-labeled alanine and the long helical axis of the was assumed to be 56°. (D) Simulation of the faster two-site methyl group jumping along the C_β-C_γ bond axis of CD₃-Leu that generates a bell-shaped spectrum and decreases the quadrupolar splitting (Δν). In panel D, the methyl group was tilted by 75° and jumped by 109.5°.

Figure 5

¹⁵N NMR powder pattern spectra of 4 mol % uniformly ¹⁵N-labeled WT-PLB (A) as well as site-specific ¹⁵N-labeled amide WT-PLB [Leu51 (B), Leu42 (C), and Ala11 (D)] and P-PLB [Ala11 (E)] proteins incorporated into POPC bilayers at 25 °C. The simulated spectra (A, D, and E) were generated by summation of its two components. The simulated spectra (B and C) were generated using one component.