Polyphenols are bioactive natural products that appear to act against a wide range of pathologies. Mechanisms of activity have not been established, but recent studies have suggested that some polyphenols bind to membranes. We examined the interaction between lipid bilayers and three structurally diverse polyphenols. We hypothesized that features of the polyphenols such as polarity, molecular size, molecular geometry, and number and arrangement of phenol hydroxyl groups would determine the tendency to interact with the bilayer. We examined a mixed polyphenol, (−) epigallocatechin gallate (EGCg); a proanthocyanidin trimer comprising catechin-(4→8)-catechin-(4→8)-catechin (cat3); and a hydrolysable tannin, 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (PGG). These polyphenols were incorporated at different levels into 2H labeled 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) multi-lamellar vesicles (MLVs). 31P and 2H solid-state NMR experiments were performed to determine the dynamics of the headgroup region and the hydrophobic acyl chain region of the lipid bilayer upon addition of polyphenols. The chemical shift anisotropy (CSA) width of the 31P NMR spectra decreased upon addition of polyphenols. Addition of PGG induces a dramatic reduction on the CSA width compared with the control lipid bilayer sample, while addition of cat3 barely reduces the CSA width. The 2H quadupolar splitting of the lipids also decreased upon addition of polyphenols. At the same concentration, PGG substantially reduced the quadrupolar splitting while cat3 barely reduced it when compared with the control sample. By calculating the order parameters of the acyl chain region of the lipid bilayer, we concluded that the hydrophobic part of the lipid bilayer was perturbed by PGG while cat3 did not cause large perturbations. The data suggest that the polarity of the polyphenols affects the interaction between tannins and membranes. The interactions may relate to the biological activities of polyphenols.

For the first time, 15 N solid-state NMR experiments were conducted on wild-type phospholamban (WT-PLB) embedded inside mechanically oriented phospholipid bilayers to investigate the topology of its cytoplasmic and transmembrane domains.15 N solid-state NMR spectra of site-specific 15 N-labeled WT-PLB indicate that the transmembrane domain has a tilt angle of 13°6 6°with respect to the POPC (1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine) bilayer normal and that the cytoplasmic domain of WT-PLB lies on the surface of the phospholipid bilayers. Comparable results were obtained from sitespecific 15 N-labeled WT-PLB embedded inside DOPC/DOPE (1,2-dioleoyl-sn-glycero-3-phosphocholine/ 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) mechanically oriented phospholipids' bilayers. The new NMR data support a pinwheel geometry of WT-PLB, but disagree with a bellflower structure in micelles, and indicate that the orientation of the cytoplasmic domain of the WT-PLB is similar to that reported for the monomeric AFA-PLB mutant.

2 H and 15 N 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. 2 H NMR spectra of site-specific CD 3 -labeled WT-PLB (at Leu51, Ala24, and Ala15) in POPC bilayers were similar under frozen conditions (-25 °C). However, significant differences in the line shapes of the 2 H NMR spectra were observed in the liquid crystalline phase at and above 0°C. The 2 H 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 2 H NMR spectrum of CD 3 -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. 2 H NMR spectra of both WT-PLB and P-PLB with CD 3 -Ala15 exhibit strong isotropic spectral line shapes. The dynamic isotropic nature of the 2 H peak can be attributed to side chain and backbone motions to residues located in an aqueous environment outside the membrane. Also, the spectra of 15 N-labeled amide WT-PLB at Leu51 and Leu42 residues showed only a single powder pattern component indicating that these two 15 N-labeled residues located in the TM helix are motionally restricted at 25 °C. Conversely, 15 N-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 2 H and 15 N NMR data indicate significant backbone motion for the cytoplasmic domain of WT-PLB when compared to the transmembrane section.Phospholamban (PLB) 1 is a 52-amino acid transmembrane protein that interacts with the CaATPase pump and lowers its affinity for Ca 2+ (1-3). PLB plays a major role in the regulation process of the cardiac cycle (contraction and relaxation), which controls the heartbeat (3-5). Unphosphorylated PLB inhibits sarcoplasmic reticulum ATPase activity and stops the flow of Ca 2+ ions, and this inhibition can be relieved by the cyclic AMP-and calmodulin-dependent phosphorylation of PLB (3-5). Since PLB is biologically significant and it is relatively small, many theoretical and biophysical experimental studies have aimed to investigate its structure in a membrane (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). † This work was supported by an AHA grant (0755602B) and a NIH Grant (GM080542). The 500 MHz wide bore NMR spectrometer was obtained from NSF Grant (10116333). NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptOn the basis of spectroscopic techniques and molecular modeling studies on pentameric WT-PLB, early structural reports on WT-PLB disagreed about whether the pentameric protein is composed of continuous α-helical subunits or composed of ...

Phospholamban (PLB) is an integral membrane protein regulating Ca2+ transport through inhibitory interaction with sarco(endo)plasmic reticulum calcium ATPase (SERCA). The Asn27 to Ala (N27A) mutation of PLB has been shown to function as a superinhibitor of the affinity of SERCA for Ca2+ and of cardiac contractility in vivo. The effects of this N27A mutation on the side-chain and backbone dynamics of PLB were investigated with 2H and 15N solid-state NMR spectroscopy in phospholipid multilamellar vesicles (MLVs). 2H and 15N NMR spectra indicate that the N27A mutation does not significantly change the side-chain or backbone dynamics of the transmembrane and cytoplasmic domains when compared to wild-type PLB. However, dynamic changes are observed for the hinge region, in which greater mobility is observed for the CD3-labeled Ala24 N27A-PLB. The increased dynamics in the hinge region of PLB upon N27A mutation may allow the cytoplasmic helix to more easily interact with the Ca2+-ATPase; thus, showing increased inhibition of Ca2+-ATPase.

The HIV-1 envelope glycoprotein gp41 fusion intermediate is a promising drug target for inhibiting viral entry. However, drug development has been impeded by challenges inherent in mediating the underlying protein-protein interaction. Here we report on the identification of fragments that bind to a C-terminal sub-pocket adjacent to the well-known hydrophobic pocket on the NHR coiled coil. Using a specifically designed assay and ligand-based NMR screening of a fragment library, we identified a thioenylaminopyrazole compound with a dissociation constant of ∼500μM. Interaction with the C-terminal sub-pocket was confirmed by paramagnetic relaxation enhancement NMR experiments, which also yielded the binding mode. Shape-based similarity searching detected additional phenylpyrazole and phenyltriazole fragments within the library, enriching the hit rate over random screening, and revealing molecular features required for activity. Discovery of the novel scaffolds and binding mechanism suggests avenues for extending the interaction surface and improving the potency of a hydrophobic pocket binding inhibitor.