Investigating the dynamic properties of the transmembrane segment of phospholamban incorporated into phospholipid bilayers utilizing 2H and 15N solid-state NMR spectroscopy.

(2)H and (15)N 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 Ca(2+)-regulator protein. (2)H NMR spectra of (2)H-labeled leucine (deuterated at one terminal methyl group) incorporated at different sites (CD(3)-Leu28, CD(3)-Leu39, and CD(3)-Leu51) along the TM-PLB peptide exhibited line shapes characteristic of either methyl group reorientation about the C(gamma)-C(delta) bond axis or by additional librational motion about the C(alpha)-C(beta) and C(beta)-C(gamma) bond axes. The (2)H NMR line shapes of all CD(3)-labeled leucines are very similar below 0 degrees 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 C(alpha)-C(beta), C(beta)-C(gamma), and C(gamma)-C(delta) bond axes as indicated by (2)H line-shape simulations and reduced quadrupolar splittings. At all of the temperatures studied, the (2)H 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 (15)N powder spectra of Leu39 and Leu42 residues indicated no backbone motion, while Leu28 exhibited slight backbone motion. The chemical-shift anisotropy tensor values for (15)N-labeled Leu TM-PLB were sigma(11) = 50.5 ppm, sigma(22) = 80.5 ppm, and sigma(33) = 229 ppm within +/-3 ppm experimental error. The (15)N chemical-shift value from the mechanically aligned spectrum of (15)N-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.