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

H-2 and N-15 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. H-2 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 H-2 NMR spectra were observed in the liquid crystalline phase at and above 0 degrees C. The H-2 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 H-2 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. H-2 NMR spectra of both WT-PLB and P-PLB with CD3-Ala15 exhibit strong isotropic spectral line shapes. The dynamic isotropic nature of the H-2 peak can be attributed to side chain and backbone motions to residues located in an aqueous environment outside the membrane. Also, the spectra of N-15-labeled amide WT-PLB at Leu51 and Leu42 residues showed only a single powder pattern component indicating that these two N-15-labeled residues located in the TM helix are motionally restricted at 25 degrees C. Conversely, N-15-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 H-2 and N-15 NMR data indicate significant backbone motion for the cytoplasmic domain of WT-PLB when compared to the transmembrane section.