Topology of the Trans-Membrane Peptide WALP23 in Model Membranes under Negative Mismatch Conditions

Matalon, Erez; Kaminker, Ilia; Zimmermann, Herbert; Eisenstein, Miriam; Shai, Yechiel; Goldfarb, Daniella

doi:10.1021/jp310056h

Cited by 16 publications

(26 citation statements)

References 76 publications

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“…The I( 2 H) values are also similar to those of residues at positions 4 and 20 in the trans-membrane a-helical peptide WALP23, which is 23 amino acid long. [16] In this case, spin labels located at the center of the WALP23 peptides revealed no modulation from D 2 O. The fact that the two ends of CP15 have comparable modulation and that a 15 amino acid helix is too short to cross the membrane support our assignment as a surface-parallel helix.…”

Section: Eseem Measurementssupporting

confidence: 52%

“…[14][15][16] The CP sequence was extended by three residues at each end ("CP15"), C-terminal amidated, and was spin-labeled by substitution with nitroxide-labeled cysteine residues at several positions . This was done using standard site-directed spin labeling (SDSL) with 1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl methanethiosulfonate (MTSSL) .…”

Section: Introductionmentioning

confidence: 99%

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The Topology, in Model Membranes, of the Core Peptide Derived from the T‐Cell Receptor Transmembrane Domain

et al. 2013

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The T-cell receptor-CD3 complex (TCR-CD3) serves a critical role in protecting organisms from infectious agents. The TCR is a heterodimer composed of α- and β-chains, which are responsible for antigen recognition. Within the transmembrane domain of the α-subunit, a region has been identified to be crucial for the assembly and function of the TCR. This region, termed core peptide (CP), consists of nine amino acids (GLRILLLKV), two of which are charged (lysine and arginine) and are crucial for the interaction with CD3. Earlier studies have shown that a synthetic peptide corresponding to the CP sequence can suppress the immune response in animal models of T-cell-mediated inflammation, by disrupting proper assembly of the TCR. As a step towards the understanding of the source of the CP activity, we focused on CP in egg phosphatidylcholine/cholesterol (9:1, mol/mol) model membranes and determined its secondary structure, oligomerization state, and orientation with respect to the membrane. To achieve this goal, 15-residue segments of TCRα, containing the CP, were synthesized and spin-labeled at different locations with a nitroxide derivative. Electron spin-echo envelope modulation spectroscopy was used to probe the position and orientation of the peptides within the membrane, and double electron-electron resonance measurements were used to probe its conformation and oligomerization state. We found that the peptide is predominantly helical in a membrane environment and tends to form oligomers (mostly dimers) that are parallel to the membrane plane.

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Section: Eseem Measurementssupporting

confidence: 52%

Section: Introductionmentioning

confidence: 99%

The Topology, in Model Membranes, of the Core Peptide Derived from the T‐Cell Receptor Transmembrane Domain

et al. 2013

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“…The 3P-ESEEM time-domain traces of DPPC-d 9 samples were simulated using a home-written MATLAB script, described in [45], based on a model presented earlier [37]. Briefly, the deuterated head group area is described as a cylinder with a radius ρ max and a height a containing N homogenously distributed 2 H nuclei, and the unpaired electron is located at a distance Z 0 from the bottom of the cylinder.…”

Section: Simulations Of the 3p-eseem Tracesmentioning

confidence: 99%

“…Simulations of the 3P-ESEEM yield two best-fit parameters, Z 0 and the 2 H nuclei density d in the layer, which is a function of N and the fixed dimensions of the cylinder. The radius of the cylinder section, whose distance from the spin label is within the range detectable by ESEEM, ρ max = 40 Å , was found from demands on integration convergence described in [45] and a = 9 Å was taken from the literature [46].…”

Section: Simulations Of the 3p-eseem Tracesmentioning

confidence: 99%

Membrane curvature and cholesterol effects on lipids packing and spin-labelled lipids conformational distributions

et al. 2013

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Nitroxide spin-labelled lipid analogues are often used to study model membrane properties using EPR spectroscopy. Whereas in liquid phase membranes the spin label assumes, on average, its putative location, in gel phases and frozen membrane, depending on its position along the acyl chain, it may exhibit a different average location. Here we used 2 H three-pulse Electron Spin Echo Envelope Modulation (ESEEM) of phospholipid spin probes, combined with various deuteration schemes to detect the effect of the model membrane curvature and cholesterol on vertical migrations of the spin label. We compared large and small unilamellar 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles with and without cholesterol (10%). The vertical displacement of the spin label was manifested as an apparently flat trans-membrane profile of water concentration and of label proximity to the head group choline. The spin-label propensity to migrate was found to increase with vesicle curvature and decrease in the presence of cholesterol. This in turn reflects the effect of packing and ordering of the membrane lipids. The results show that in curved vesicles lacking cholesterol, the label attached to carbon 16 may travel as far high along the membrane normal as the location of the label on carbon 5, due to the presence of U-shaped lipid conformations. This phenomenon must be taken into account when using spin-labelled lipids as membrane depth markers or to trace trans-membrane profiles.

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“…In the past decade, EPR distance measurements have emerged as an important technique to characterize structure in membrane proteins (Altenbach et al, 2008;Georgieva et al, 2013;Hilger et al, 2005;Jeschke, 2013a;Matalon et al, 2013b;McHaourab et al, 2011). EPR allows the precise measurement of distances between spin labels attached to proteins or other biomolecules (Berliner et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

Determining the Oligomeric Structure of Proteorhodopsin by Gd3+-Based Pulsed Dipolar Spectroscopy of Multiple Distances

et al. 2014

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The structural organization of the functionally relevant, hexameric oligomer of green-absorbing proteorhodopsin (G-PR) was obtained from double electron-electron resonance (DEER) spectroscopy utilizing conventional nitroxide spin labels and recently developed Gd3+ -based spin labels. G-PR with nitroxide or Gd3+ labels was prepared using cysteine mutations at residues Trp58 and Thr177. By combining reliable measurements of multiple interprotein distances in the G-PR hexamer with computer modeling, we obtained a structural model that agrees with the recent crystal structure of the homologous blue-absorbing PR (B-PR) hexamer. These DEER results provide specific distance information in a membrane-mimetic environment and across loop regions that are unresolved in the crystal structure. In addition, the X-band DEER measurements using nitroxide spin labels suffered from multispin effects that, at times, compromised the detection of next-nearest neighbor distances. Performing measurements at high magnetic fields with Gd3+ spin labels increased the sensitivity considerably and alleviated the difficulties caused by multispin interactions.

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Topology of the Trans-Membrane Peptide WALP23 in Model Membranes under Negative Mismatch Conditions

Cited by 16 publications

References 76 publications

The Topology, in Model Membranes, of the Core Peptide Derived from the T‐Cell Receptor Transmembrane Domain

The Topology, in Model Membranes, of the Core Peptide Derived from the T‐Cell Receptor Transmembrane Domain

Membrane curvature and cholesterol effects on lipids packing and spin-labelled lipids conformational distributions

Determining the Oligomeric Structure of Proteorhodopsin by Gd3+-Based Pulsed Dipolar Spectroscopy of Multiple Distances

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