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The solid state NMR spectroscopy was used for (super 31)P and (super 2)H relaxation studies of helix VI and the cytoplasmic helix of human cannabinoid receptor. This technique can be used to sense the physical state of the lipid bilayer as well as the molecular motions associated with acyl chains in the presence of macromolecules.

The dynamics of site-specific-labeled CD3 groups of leucine in TM-PLB as a function of temperature is probed. The solid-state NMR data presented on TM-PLB does not specifically favor either of the two structural models for the full-length PLB pentamer.

A novel electron paramagnetic resonance (EPR) spectroscopy method is developed to study the spin-labeled peptides inside aligned lipid bilayer nanotube arrays. The rigidly coupled TOAC spin label is used to determine the helical tilt of the peptide with respect to the membrane.

A transmembrane peptide containing the aminoproximate TM-A domain of the integral membrane acetylenase CREP-1 is used to illustrate the feasibility of using phospholipid bilayer nanotube arrays for solid-state NMR studies on integral membrane proteins. Studies on the integral membraneprotein TM-A indicate that the peptide is dominantly alpha-helical.

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]

Abstract: Solid-state NMR spectroscopic techniques were used to investigate the secondary structure of the transmembrane peptide phospholamban (TM-PLB), a sarcoplasmic Ca2+ regulator. 13C cross-polarization magic angle spinning spectra of 13C carbonyl-labeled Leu39 of TM-PLB exhibited two peaks in a pure 1-palmitoyl-2-oleoyl-phosphocholine (POPC) bilayer, each due to a different structural conformation of phospholamban as characterized by the corresponding 13C chemical shift. The addition of a negatively charged phospholipid (1-palmitoyl-2-oleoylphosphatidylglycerol (POPG)) to the POPC bilayer stabilized TM-PLB to an α-helical conformation as monitored by an enhancement of the α-helical carbonyl 13C resonance in the corresponding NMR spectrum. 13C–15N REDOR solid-state NMR spectroscopic experiments revealed the distance between the 13C carbonyl carbon of Leu39 and the 15N amide nitrogen of Leu42 to be 4.2±0.2Å indicating an α-helical conformation of TM-PLB with a slight deviation from an ideal 3.6 amino acid per turn helix. Finally, the quadrupolar splittings of three 2H labeled leucines (Leu28, Leu39, and Leu51) incorporated in mechanically aligned DOPE/DOPC bilayers yielded an 11°±5° tilt of TM-PLB with respect to the bilayer normal. In addition to elucidating valuable TM-PLB secondary structure information, the solid-state NMR spectroscopic data indicates that the type of phospholipids and the water content play a crucial role in the secondary structure and folding of TM-PLB in a phospholipid bilayer. [Copyright &y& Elsevier]

²H and 15N 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 Ca2+-regulator protein. ²H NMR spectra of ²H-labeled leucine (deuterated at one terminal methyl group) incorporated at different sites (CD3-Leu28, CD3-Leu39, and CD3-Leu51) along the TM-PLB peptide exhibited line shapes characteristic of either methyl group reorientation about the Cγ-Cδ bond axis or by additional librational motion about the Cα-Cβ and Cβ-Cγ bond axes. The ²H NMR line shapes of all CD3-labeled leucines are very similar below 0°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 Cα-Cβ, Cβ-Cγ and Cγ-Cδ bond axes as indicated by ²H line-shape simulations and reduced quadrupolar splittings. At all of the temperatures studied, the ²H 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 15N powder spectra of Leu39 and Leu42 residues indicated no backbone motion, while Leu28 exhibited slight backbone motion. The chemical- shift anisotropy tensor values for 15N-labeled Leu TM-PLB were σ11 = 50.5 ppm, σ22 = 80.5 ppm, and σ33 = 229 ppm within ±3 ppm experimental error. The 15N chemical-shift value from the mechanically aligned spectrum of 15N-labeled Leu39 PLB in DOPCIDOPE phospholipid bilayers was 220 ppm and is characteristic of a TM peptide that is nearly parallel with the bilayer normal. [ABSTRACT FROM AUTHOR]

PDZ domains are independently folded modules that typically mediate protein-protein interactions by binding to the C termini of their target proteins. However, in a few instances, PDZ domains have been reported to dimerize with other PDZ domains. To investigate this noncanonical-binding mode further, we used protein microarrays comprising virtually every mouse PDZ domain to systematically query all possible PDZ-PDZ pairs. We then used fluorescence polarization to retest and quantify interactions and coaffinity purification to test biophysically validated interactions in the context of their full-length proteins. Overall, we discovered 37 PDZ-PDZ interactions involving 46 PDZ domains (~30% of all PDZ domains tested), revealing that dimerization is a more frequently used binding mode than was previously appreciated. This suggests that many PDZ domains evolved to form multiprotein complexes by simultaneously interacting with more than one ligand., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

This study reports the solid-state NMR spectroscopic characterization of the amino-proximate transmembrane domain (TM-A) of a diverged microsomal delta12-desaturase (CREP-1) in a phospholipid bilayer. A series of TM-A peptides were synthesized with 2H-labeled side chains (Ala-53, -56, and -63, Leu-62, Val-50), and their dynamic properties were studied in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) bilayers at various temperatures. At 6 mol % peptide to lipid, 31P NMR spectra indicated that the peptides did not significantly disrupt the phospholipid bilayer in the L(alpha) phase. The 2H NMR spectra from Ala-53 and Ala-56 samples revealed broad Pake patterns with quadrupolar splittings of 16.9 kHz and 13.3 kHz, respectively, indicating restricted motion confined within the hydrocarbon core of the phospholipid bilayer. Conversely, the deuterated Ala-63 sample revealed a peak centered at 0 kHz with a linewidth of 1.9 kHz, indicating increased side-chain motion and solvent exposure relative to the spectra of the other Ala residues. Val-50 and Leu-62 showed Pake patterns, with quadrupolar splittings of 3.5 kHz and 3.7 kHz, respectively, intermediate to Ala-53/Ala-56 and Ala-63. This indicates partial motional averaging and supports a model with the Val and Leu residues embedded inside the lipid bilayer. Solid-state NMR spectroscopy performed on the 2H-labeled Ala-56 TM-A peptide incorporated into magnetically aligned phospholipid bilayers indicated that the peptide is tilted 8 degrees with respect to the membrane normal of the lipid bilayer. Snorkeling and anchoring interactions of Arg-44 and Tyr-60, respectively, with the polar region or polar hydrophobic interface of the lipid bilayer are suggested as control elements for insertional depth and orientation of the helix in the lipid matrix. Thus, this study defines the location of key residues in TM-A with respect to the lipid bilayer, describes the conformation of TM-A in a biomembrane mimic, presents a peptide-bilayer model useful in the consideration of local protein folding in the microsomal desaturases, and presents a model of arginine and tyrosine control of transmembrane protein stability and insertion.