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Four new swapped-domain constructs of the ectodomain of human immunodeficiency virus type 1 glycoprotein-41 (gp41) were prepared. The gp41 ectodomain consists of 50-residue N-heptad repeat (NHR), 36-residue disulfide-bonded loop and 39-residue C-heptad repeat (CHR). It folds into a hairpin structure that forms a trimer along the NHR axis. The swapped-domain proteins feature CHR domains of length 39, 28 or 21 residues preceding a 4-residue loop and a 49- or 50-residue NHR domain. The effect of CHR truncation was to expose increasing lengths of the NHR groove, including the conserved hydrophobic pocket, an important drug target. A novel method for preparing proteins with extended exposed hydrophobic surfaces was demonstrated. Biophysical measurements, including analytical ultracentrifugation and ligand-detected Water-Ligand Observed via Gradient Spectroscopy and ¹H-15N-HSQC NMR experiments, were used to confirm that the proteins formed stable trimers in solution with exposed binding surfaces. These proteins could play an important role as receptors in structure-based drug discovery. [ABSTRACT FROM AUTHOR]

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]

Cannabinoid receptors are G-protein-coupled receptors comprised of seven transmembrane helices. We hypothesized that the extended helix of the receptor interacts differently with POPC bilayers due to the differing distribution of charged amino acid residues. To test this, hCB1(T377—E416) and hCB2(K278—H316) peptides were studied with 31P and 2H solid-state NMR spectroscopy by incorporating them into 1-palmitoyl-2-oleoyl-sn-glycerophosphocholine bilayers. Lipid affinities of the 40- and 39- residue peptides were analyzed on the basis of 31P and 2H spectral line shapes, order parameters, and T1 relaxation measurements of the POPC bilayers. Lipid headgroup perturbations were noticed in the 31P NMR spectra in the lipid/peptide mixtures when compared with the pure lipids. 2H order parameters were calculated from the quadrupolar splitting of the de-Paked 2H NMR spectra. At the top of the acyl chain, pure lipids had an average SCD ≈ 0.20, whereas SCD ≈ 0.16 and SCD ≈ 0.18 were found in the presence of hCB1 (T377–E416) and hCB2(K278–H316), respectively. SCD values decreased in the central part of the acyl chains when compared to the pure POPC lipids, indicating a change in the dynamic properties of the lipid membrane in the presence of the cannabinoid peptides. R1z vs S2CD plots exhibited a linear dependency with and without the peptides, with an increase in slope upon addition of the peptides to the POPC, indicating that the dynamics of the lipid bilayer is dominated by fast axially symmetric motion. This study provides insights into the interaction of cannabinoid peptides with the membrane bilayer by investigating the headgroup and acyl chain dynamics. [ABSTRACT FROM AUTHOR]