Family-wide investigation of PDZ domain-mediated protein-protein interactions implicates ß-catenin in maintaining the integrity of tight junctions.

ß-catenin is a multifunctional protein that plays a critical role in cell-cell contacts and signal transduction. ß-catenin has previously been shown to interact with PDZ-domain-containing proteins through its C terminus. Using protein microarrays comprising 206 mouse PDZ domains, we identified 26 PDZ-domain-mediated interactions with ß-catenin and confirmed them biochemically and in cellular lysates. Many of the previously unreported interactions involved proteins with annotated roles in tight junctions. We found that four tight-junction-associated PDZ proteins-Scrib, Magi-1, Pard3, and ZO-3-colocalize with ß-catenin at the plasma membrane. Disrupting these interactions by RNA interference, overexpression of PDZ domains, or overexpression of the ß-catenin C terminus altered localization of the full-length proteins, weakened tight junctions, and decreased cellular adhesion. These results suggest that ß-catenin serves as a scaffold to establish the location and function of tight-junction-associated proteins.

Time-resolved EPR immersion depth studies of a transmembrane peptide incorporated into bicelles.

The reduction in EPR signal intensity of nitroxide spin-labels by ascorbic acid has been measured as a function of time to investigate the immersion depth of the spin-labeled M2δ AChR peptide incorporated into a bicelle system utilizing EPR spectroscopy. The corresponding decay curves of n-DSA (n=5, 7, 12, and 16) EPR signals have been used to (1) calibrate the depth of the bicelle membrane and (2) establish a calibration curve for measuring the depth of spin-labeled transmembrane peptides. The kinetic EPR data of CLS, n-DSA (n=5, 7, 12, and 16), and M2δ AChR peptide spin-labeled at Glu-1 and Ala-12 revealed excellent exponential and linear fits. For a model M2δ AChR peptide, the depth of immersion was calculated to be 5.8Å and 3Å for Glu-1, and 21.7Å and 19Å for Ala-12 in the gel-phase (298K) and L(α)-phases (318K), respectively. The immersion depth values are consistent with the pitch of an α-helix and the structural model of M2δ AChR incorporated into the bicelle system is in a good agreement with previous studies. Therefore, this EPR time-resolved kinetic technique provides a new reliable method to determine the immersion depth of membrane-bound peptides, as well as, explore the structural characteristics of the M2δ AChR peptide.

A systematic family-wide investigation reveals that ~30% of mammalian PDZ domains engage in PDZ-PDZ interactions.

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.

Quantifying protein-protein interactions in high throughput using protein domain microarrays.

Protein microarrays provide an efficient way to identify and quantify protein-protein interactions in high throughput. One drawback of this technique is that proteins show a broad range of physicochemical properties and are often difficult to produce recombinantly. To circumvent these problems, we have focused on families of protein interaction domains. Here we provide protocols for constructing microarrays of protein interaction domains in individual wells of 96-well microtiter plates, and for quantifying domain-peptide interactions in high throughput using fluorescently labeled synthetic peptides. As specific examples, we will describe the construction of microarrays of virtually every human Src homology 2 (SH2) and phosphotyrosine binding (PTB) domain, as well as microarrays of mouse PDZ domains, all produced recombinantly in Escherichia coli. For domains that mediate high-affinity interactions, such as SH2 and PTB domains, equilibrium dissociation constants (K(D)s) for their peptide ligands can be measured directly on arrays by obtaining saturation binding curves. For weaker binding domains, such as PDZ domains, arrays are best used to identify candidate interactions, which are then retested and quantified by fluorescence polarization. Overall, protein domain microarrays provide the ability to rapidly identify and quantify protein-ligand interactions with minimal sample consumption. Because entire domain families can be interrogated simultaneously, they provide a powerful way to assess binding selectivity on a proteome-wide scale and provide an unbiased perspective on the connectivity of protein-protein interaction networks.

Vertical silicon nanowires as a universal platform for delivering biomolecules into living cells.

A generalized platform for introducing a diverse range of biomolecules into living cells in high-throughput could transform how complex cellular processes are probed and analyzed. Here, we demonstrate spatially localized, efficient, and universal delivery of biomolecules into immortalized and primary mammalian cells using surface-modified vertical silicon nanowires. The method relies on the ability of the silicon nanowires to penetrate a cell's membrane and subsequently release surface-bound molecules directly into the cell's cytosol, thus allowing highly efficient delivery of biomolecules without chemical modification or viral packaging. This modality enables one to assess the phenotypic consequences of introducing a broad range of biological effectors (DNAs, RNAs, peptides, proteins, and small molecules) into almost any cell type. We show that this platform can be used to guide neuronal progenitor growth with small molecules, knock down transcript levels by delivering siRNAs, inhibit apoptosis using peptides, and introduce targeted proteins to specific organelles. We further demonstrate codelivery of siRNAs and proteins on a single substrate in a microarray format, highlighting this technology's potential as a robust, monolithic platform for high-throughput, miniaturized bioassays.

Characterization of lipid bilayer formation in aligned nanoporous aluminum oxide nanotube arrays.

Aligning lipid bilayers in nanoporous anodized aluminum oxide (AAO) is a new method to help study membrane proteins by electron paramagnetic resonance (EPR) and solid-state nuclear magnetic resonance (NMR) spectroscopic methods. The ability to maintain hydration, sample stability, and compartmentalization over long periods of time, and to easily change solvent composition are major advantages of this new method. To date, 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) has been the only phospholipid used for membrane protein studies with AAO substrates. The different properties of lipids with varying chain lengths require modified sample preparation procedures to achieve well formed bilayers within the lining of the AAO substrates. For the first time, the current study presents a simple methodology to incorporate large quantities of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), DMPC, and 1,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC) phospholipids inside AAO substrate nanopores of varying sizes. (2)H and (31)P solid-state NMR were used to confirm the alignment of each lipid and compare the efficiency of alignment. This study is the first step in standardizing the use of AAO substrates as a tool in NMR and EPR and will be useful for future structural studies of membrane proteins. Additionally, the solid-state NMR data suggest possible applications of nanoporous aluminum oxide in future vesicle fusion studies.

Electron paramagnetic resonance studies of an integral membrane peptide inserted into aligned phospholipid bilayer nanotube arrays.

This communication reports for the first time the determination of the helical tilt of an integral membrane peptide inserted into aligned phospholipids bilayer nanotube arrays using spin label EPR spectroscopy. Also, we demonstrate herein how the helical tilt of the peptide can be easily calculated using the hyperfine splitting values gleaned from a perpendicularly aligned bilayer in phospholipid bilayer nanotube arrays. EPR spectral simulations were used to verify the method.

The structural properties of the transmembrane segment of the integral membrane protein phospholamban utilizing (13)C CPMAS, (2)H, and REDOR solid-state NMR spectroscopy.

Solid-state NMR spectroscopic techniques were used to investigate the secondary structure of the transmembrane peptide phospholamban (TM-PLB), a sarcoplasmic Ca(2+) regulator. (13)C cross-polarization magic angle spinning spectra of (13)C 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 (13)C chemical shift. The addition of a negatively charged phospholipid (1-palmitoyl-2-oleoylphosphatidylglycerol (POPG)) to the POPC bilayer stabilized TM-PLB to an alpha-helical conformation as monitored by an enhancement of the alpha-helical carbonyl (13)C resonance in the corresponding NMR spectrum. (13)C-(15)N REDOR solid-state NMR spectroscopic experiments revealed the distance between the (13)C carbonyl carbon of Leu39 and the (15)N amide nitrogen of Leu42 to be 4.2+/-0.2A indicating an alpha-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 (2)H labeled leucines (Leu28, Leu39, and Leu51) incorporated in mechanically aligned DOPE/DOPC bilayers yielded an 11 degrees +/-5 degrees 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.

31P and 2H relaxation studies of helix VII and the cytoplasmic helix of the human cannabinoid receptors utilizing solid-state NMR techniques.

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 S(CD) approximately = 0.20, whereas S(CD) approximately = 0.16 and S(CD) approximately = 0.18 were found in the presence of hCB1(T377-E416) and hCB2(K278-H316), respectively. S(CD) 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. R(1Z) vs S2(CD) 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.

Solid-state NMR studies of a diverged microsomal amino-proximate delta12 desaturase peptide reveal causes of stability in bilayer: tyrosine anchoring and arginine snorkeling.

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.

Investigating the dynamic properties of the transmembrane segment of phospholamban incorporated into phospholipid bilayers utilizing 2H and 15N solid-state NMR spectroscopy.

(2)H and (15)N 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 Ca(2+)-regulator protein. (2)H NMR spectra of (2)H-labeled leucine (deuterated at one terminal methyl group) incorporated at different sites (CD(3)-Leu28, CD(3)-Leu39, and CD(3)-Leu51) along the TM-PLB peptide exhibited line shapes characteristic of either methyl group reorientation about the C(gamma)-C(delta) bond axis or by additional librational motion about the C(alpha)-C(beta) and C(beta)-C(gamma) bond axes. The (2)H NMR line shapes of all CD(3)-labeled leucines are very similar below 0 degrees 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 C(alpha)-C(beta), C(beta)-C(gamma), and C(gamma)-C(delta) bond axes as indicated by (2)H line-shape simulations and reduced quadrupolar splittings. At all of the temperatures studied, the (2)H 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 (15)N powder spectra of Leu39 and Leu42 residues indicated no backbone motion, while Leu28 exhibited slight backbone motion. The chemical-shift anisotropy tensor values for (15)N-labeled Leu TM-PLB were sigma(11) = 50.5 ppm, sigma(22) = 80.5 ppm, and sigma(33) = 229 ppm within +/-3 ppm experimental error. The (15)N chemical-shift value from the mechanically aligned spectrum of (15)N-labeled Leu39 PLB in DOPC/DOPE phospholipid bilayers was 220 ppm and is characteristic of a TM peptide that is nearly parallel with the bilayer normal.

Solid-state NMR spectroscopic studies of an integral membrane protein inserted into aligned phospholipid bilayer nanotube arrays.

This communication demonstrates for the first time that solid-state NMR spectroscopic studies can be used to investigate aligned phospholipid bilayer nanotube arrays. Also, an integral membrane peptide can be successfully incorporated into the oriented phospholipid bilayer nanotube arrays and studied with 2H solid-state NMR spectroscopy.