Investigating the dynamic properties of the transmembrane segment of phospholamban incorporated into phospholipid bilayers utilizing ²H and ¹⁵N Solid-State NMR Spectroscopy †

²H and ¹⁵N solid-state NMR spectroscopic techniques were used to investigate the membrane composition, orientation, and side-chain dynamics of the transmembrane segment of phospholamban (TM-PLB), a sarcoplasmic Ca²⁺-regulator protein. ²H NMR spectra of ²H-labeled leucine (deuterated at one terminal methyl group) incorporated at different sites (CD₃-Leu28, CD₃-Leu39, and CD₃-Leu51) along the TM-PLB peptide exhibited line shapes characteristic of either methyl group reorientation about the Cγ-Cδ bond axis or by additional librational motion about the Cα-Cβ and Cβ-Cγ bond axes. The ²H NMR line shapes of all CD ₃-labeled leucines are very similar below 0°C, indicating that all of the residues are located inside the lipid bilayer. At higher temperatures, all three labeled leucine residues undergo rapid reorientation about the Cα-Cβ, Cβ-Cγ, and Cγ-Cδ bond axes as indicated by ²H line-shape simulations and reduced quadrupolar splittings. At all of the temperatures studied, the ²H NMR spectra indicated that the Leu51 side chain has less motion than Leu39 or Leu28, which is attributed to its incorporation in the pentameric PLB leucine zipper motif. The ¹⁵N powder spectra of Leu39 and Leu42 residues indicated no backbone motion, while Leu28 exhibited slight backbone motion. The chemical-shift anisotropy tensor values for ¹⁵N-labeled Leu TM-PLB were σ₁₁ = 50.5 ppm, σ₂₂ = 80.5 ppm, and σ₃₃ = 229 ppm within ±3 ppm experimental error. The ¹⁵N chemical-shift value from the mechanically aligned spectrum of ¹⁵N-labeled Leu39 PLB in DOPC/DOPE phospholipid bilayers was 220 ppm and is characteristic of a TM peptide that is nearly parallel with the bilayer normal.