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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 I -palmitoyl-2-oleoyl-sn-glycerophosphocholine (POPC) phosphol ipid bilayers. 2H NMR spectra of site-specific CD3-labeled WT-PLB (at Leu51, Ala24, and Ala 15) in POPC bilayers were similar under frozen conditions (-25 °C). However, significant differences in the line shapes of the 2H NMR spectra were observed in the liquid crystalline phase at and above 0 °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 °C. Conversely, 15N- labeled amide WT-PLB at Ala11 located in the cytoplasmic domain showed both powder and isotropic components at 25 °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. [ABSTRACT FROM AUTHOR]

An 18-residue peptide, KWGAKIKIGAKIKIGAKI-NH2 was designed to form amphiphilic β-sheet structures when bound to lipid bilayers. The peptide possesses high antimicrobial activity when compared to naturally occurring linear antimicrobial peptides, most of which adopt an amphipathic α-helical conformation upon binding to the lipids. The perturbation of the bilayer by the peptide was studied by static 31P and 2H solid-state NMR spectroscopy using POPC and POPG/POPC (3/1) bilayer membranes with sn-1 chain perdeuterated POPC and POPG as the isotopic labels. 31P NMR powder spectra exhibited two components for POPG/POPC bilayers upon addition of the peptide but only a slight change in the line shape for POPC bilayers, indicating that the peptide selectively disrupted the membrane structure consisting of POPG lipids. 2H NMR powder spectra indicated a reduction in the lipid chain order for POPC bilayers and no significant change in the ordering for POPG/POPC bilayers upon association of the peptide with the bilayers, suggesting that the peptide acts as a surface peptide in POPG/POPC bilayers. Relaxation rates are more sensitive to the motions of the membranes over a large range of time scales. Longer 31P longitudinal relaxation times for both POPG and POPC in the presence of the peptide indicated a direct interaction between the peptide and the POPG/POPC bilayer membranes. 31P longitudinal relaxation studies also suggested that the peptide prefers to interact with the POPG phospholipids. However, inversion-recovery 2H NMR spectroscopic experiments demonstrated a change in the relaxation rate of the lipid acyl chains for both the POPC membranes and the POPG/POPC membranes upon interaction with the peptide. Transverse relaxation studies indicated an increase in the spectral density of the collective membrane motion caused by the interaction between the peptide and the POPG/POPC membrane. The experimental results demonstrate significant dynamic changes in the membrane in the presence of the antimicrobial peptide and support a carpet mechanism for the disruption of the membranes by the antimicrobial peptide. [ABSTRACT FROM AUTHOR]