Investigating the Dynamic Properties of the Transmembrane Segment of Phospholamban Incorporated into Phospholipid Bilayers Utilizing 2H and `5N 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 CD3-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 DOPCIDOPE phospholipid bilayers was 220 ppm and is characteristic of a TM peptide that is nearly parallel with the bilayer normal. [ABSTRACT FROM AUTHOR]