Transmembrane Helical Peptides Research Articles

Frequency Changes in Terminal Alkynes Provide Strong, Sensitive, and Solvatochromic Raman Probes of Biochemical Environments.

The C≡C stretching frequencies of terminal alkynes appear in the "clear" window of vibrational spectra, so they are attractive and increasingly popular as site-specific probes in complicated biological systems like proteins, cells, and tissues. In this work, we collected infrared (IR) absorption and Raman scattering spectra of model compounds, artificial amino acids, and model proteins that contain terminal alkyne groups, and we used our results to draw conclusions about the signal strength and sensitivity to the local environment of both aliphatic and aromatic terminal alkyne C≡C stretching bands. While the IR bands of alkynyl model compounds displayed surprisingly broad solvatochromism, their absorptions were weak enough that alkynes can be ruled out as effective IR probes. The same solvatochromism was observed in model compounds' Raman spectra, and comparisons to published empirical solvent scales (including a linear regression against four meta-aggregated solvent parameters) suggested that the alkyne C≡C stretching frequency mainly reports on local electronic interactions (i.e., short-range electron donor-acceptor interactions) with solvent molecules and neighboring functional groups. The strong solvatochromism observed here for alkyne stretching bands introduces an important consideration for Raman imaging studies based on these signals. Raman signals for alkynes (especially those that are π-conjugated) can be exceptionally strong and should permit alkynyl Raman signals to function as probes at very low concentrations, as compared to other widely used vibrational probe groups like azides and nitriles. We incorporated homopropargyl glycine into a transmembrane helical peptide via peptide synthesis, and we installed p-ethynylphenylalanine into the interior of the Escherichia coli fatty acid acyl carrier protein using a genetic code expansion technique. The Raman spectra from each of these test systems indicate that alkynyl C≡C bands can act as effective and unique probes of their local biomolecular environments. We provide guidance for the best possible future uses of alkynes as solvatochromic Raman probes, and while empirical explanations of the alkyne solvatochromism are offered, open questions about its physical basis are enunciated.

Comparing Interfacial Trp, Interfacial His and pH Dependence for the Anchoring of Tilted Transmembrane Helical Peptides

Charged and aromatic amino acid residues, being enriched toward the terminals of membrane-spanning helices in membrane proteins, help to stabilize particular transmembrane orientations. Among them, histidine is aromatic and can be positively charge at low pH. To enable investigations of the underlying protein-lipid interactions, we have examined the effects of single or pairs of interfacial histidine residues using the constructive low-dynamic GWALP23 (acetyl-GG2ALW5LALALALALALALW19LAG22A-amide) peptide framework by incorporating individual or paired histidines at locations 2, 5, 19 or 22. Analysis of helix orientation by means of solid-state 2H NMR spectra of labeled alanine residues reveals marked differences with H2,22 compared to W2,22. Nevertheless, the properties of membrane-spanning H2,22WALP23 helices show little pH dependence and are similar to those having Gly, Arg or Lys at positions 2 and 22. The presence of H5 or H19 influences the helix rotational preference but not the tilt magnitude. H5 affects the helical integrity, as residue 7 unwinds from the core helix; yet once again the helix orientation and dynamic properties show little sensitivity to pH. The overall results reveal that the detailed properties of transmembrane helices depend upon the precise locations of interfacial histidine residues.

Membrane Bending Moduli of Coexisting Liquid Phases Containing Transmembrane Peptide

A number of highly curved membranes in vivo, such as epithelial cell microvilli, have the relatively high sphingolipid content associated with “raft-like” composition. Given the much lower bending energy measured for bilayers with “nonraft” low sphingomyelin and low cholesterol content, observing high curvature for presumably more rigid compositions seems counterintuitive. To understand this behavior, we measured membrane rigidity by fluctuation analysis of giant unilamellar vesicles. We found that including a transmembrane helical GWALP peptide increases the membrane bending modulus of the liquid-disordered (Ld) phase. We observed this increase at both low-cholesterol fraction and higher, more physiological cholesterol fraction. We find that simplified, commonly used Ld and liquid-ordered (Lo) phases are not representative of those that coexist. When Ld and Lo phases coexist, GWALP peptide favors the Ld phase with a partition coefficient of 3–10 depending on mixture composition. In model membranes at high cholesterol fractions, Ld phases with GWALP have greater bending moduli than the Lo phase that would coexist.

Sequence-nonspecific Stabilization of Transmembrane Helical Peptide Dimer in Lipid Raft-like Bilayers in Atomistic Simulations. I. Dimerization Free Energy and Impact of Lipid-peptide Potential Energy

Sequence-nonspecific Stabilization of Transmembrane Helical Peptide Dimer in Lipid Raft-like Bilayers in Atomistic Simulations. I. Dimerization Free Energy and Impact of Lipid-peptide Potential Energy

Sequence-nonspecific Stabilization of Transmembrane Helical Peptide Dimer in Lipid Raft-like Bilayers in Atomistic Simulations. II. Acyl Chain OrderAssociated Changes in Lipid-peptide Contacts Concordant with Potential Energy Profiles

Sequence-nonspecific Stabilization of Transmembrane Helical Peptide Dimer in Lipid Raft-like Bilayers in Atomistic Simulations. II. Acyl Chain OrderAssociated Changes in Lipid-peptide Contacts Concordant with Potential Energy Profiles

Be straight to better coordinate two -- Head groups and extended acyl chains of raft-like bilayer lipids can localize in groove between dimerized transmembrane helical peptides and assist sequence-nonspecific stabilization of peptide dimers by cholesterol

Be straight to better coordinate two -- Head groups and extended acyl chains of raft-like bilayer lipids can localize in groove between dimerized transmembrane helical peptides and assist sequence-nonspecific stabilization of peptide dimers by cholesterol

Influence of High pH and Cholesterol on Single Arginine-Containing Transmembrane Peptide Helices.

An essential component of mammalian cells, cholesterol exerts significant influence on the physical properties of the cell membrane and in turn its constituents, including membrane proteins. Although sparse, polar amino acid residues are highly conserved in membrane proteins and play pivotal roles in determining specific structural and functional properties. To improve our understanding of particular polar residues in the membrane environment, we have examined two specific “guest” Arg residues within a well-defined and deuterium-labeled “host” framework provided by the transmembrane helical peptide GWALP23 (acetyl-GGALWLALALALALALALWLAGA-amide). Solid-state 2H nuclear magnetic resonance (NMR) spectra from aligned bilayer membrane samples effectively report changes in the host helix properties because of the incorporation of the guest residues. The focus of this work is two-pronged. First, GWALP23-R14 was examined over a pH range of 2–13 in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) ester- or ether-linked bilayer membranes. Our findings indicate that the Arg guanidinium side chain remains charged over this entire range, in agreement with numerous molecular dynamics simulations. Second, GWALP23-R12 and GWALP23-R14 peptides were characterized in DOPC bilayers with varying cholesterol content. Our findings suggest that 10 or 20% cholesterol content has minimal impact on the orientation of the R14 peptide. Although the NMR signals are broader and weaker in the presence of 20% cholesterol, the deuterium quadrupolar splittings for [2H]Ala residues in GWALP23-R14 change very little. Conversely, cholesterol appears to modulate the multistate behavior of GWALP23-R12 and to favor a major interfacial state for the helix, bound at the bilayer surface. These results indicate a conditional sensitivity of a complex multistate transmembrane Arg-containing peptide helix to the presence of cholesterol.

Bending Moduli of Ternary Mixture Models of the Cell Plasma Membrane

Cells must be able to maintain and change membrane shape. This membrane curvature can be influenced by any lateral phase heterogeneity. Ternary mixtures of high-melting lipid, low-melting lipid, and cholesterol exhibit a region of liquid-ordered (Lo)+ liquid-disordered (Ld) phase coexistence analogous to raft + non-raft behavior in cells. Although curvature can induce separation and sorting in vitro, a number of highly-curved membranes in vivo have raft-like composition, which is puzzling since the raft-like compositions have greater rigidity in lipid model mixtures. We find that including transmembrane helical WALP peptides as a component of the model mixtures greatly changes their bending behavior. Measurements were made by shape analysis of giant unilamellar vesicles with both fluorescence and phase contrast microscopy. Vesicles of a single phase were made by the gentle hydration method to obtain a more narrow distribution of vesicle tensions than is obtained by electroformation. We find that a WALP peptide component rigidifies the liquid-disordered phase whereas the liquid-ordered phase is unaffected. This rigidifying effect might be related to the monolayers sliding relative to each other. Further, we have investigated the effects of peptides of different lengths and results suggest a role for hydrophobic mismatch of the peptide component relative to the bilayer thickness.

Free energy of helical transmembrane peptide dimerization in OPLS-AA/Berger force field simulations: inaccuracy and implications for partner-specific Lennard-Jones parameters between peptides and lipids

Interactions between transmembrane (TM) peptides are important in biophysical chemistry, but there are few studies assessing atomistic simulation parameters concerning the energetics of interactions of TM helical peptides. Our potential of mean force analysis using OPLS-AA protein/Berger lipid force fields (FFs) shows that the dimerisation energy of (AALALAA)3 helical peptides in the dioleoylphosphatidylcholine bilayer is −4.4 kJ/mol, which was much smaller than the reported experimental value (−12.7 kJ/mol), thus calling for improvement of parameters of the combined FFs. As each of the FFs has been independently developed, we then tested the effects of downscaling the Lennard-Jones (LJ) energy terms between the OPLS-AA atoms and Berger lipid atoms, preserving the parameters within each FF. A 0.9-fold rescaling of the LJ energies was found to render the dimerisation energy close to the experimental value. Solvation of backbone atoms as well as side chain atoms in lipids is crucial for the TM helix interaction. In similar analyses, GROMOS 53A6 FF exhibited as weak dimerisation propensity (~−5.2 kJ/mol) as OPLS-AA/Berger, but CHARMM36 showed relatively accurate propensity (~−9.9 kJ/mol). Challenges and strategies in rendering the TM interaction energy realistic within the framework of current FFs are discussed.

Solid-State NMR/Dynamic Nuclear Polarization of Polypeptides in Planar Supported Lipid Bilayers.

Dynamic nuclear polarization has been developed to overcome the limitations of the inherently low signal intensity of NMR spectroscopy. This technique promises to be particularly useful for solid-state NMR spectroscopy where the signals are broadened over a larger frequency range and most investigations rely on recording low gamma nuclei. To extend the range of possible investigations, a triple-resonance flat-coil solid-state NMR probe is presented with microwave irradiation capacities allowing the investigation of static samples at temperatures of 100 K, including supported lipid bilayers. The probe performance allows for two-dimensional separated local field experiments with high-power Lee-Goldberg decoupling and cross-polarization under simultaneous irradiation from a gyrotron microwave generator. Efficient cooling of the sample turned out to be essential for best enhancements and line shape and necessitated the development of a dedicated cooling chamber. Furthermore, a new membrane-anchored biradical is presented, and the geometry of supported membranes was optimized not only for good membrane alignment, handling, stability, and filling factor of the coil but also for heat and microwave dissipation. Enhancement factors of 17-fold were obtained, and a two-dimensional PISEMA spectrum of a transmembrane helical peptide was obtained in less than 2 h.

Membrane Bending Modulus for Ternary Mixture Models of the Cell Plasma Membrane

Ternary mixtures of high-melting lipid, low-melting lipid, and cholesterol exhibit a region of liquid-ordered + liquid-disordered phase coexistence analogous to raft + non-raft behavior in cells. These coexisting phases manifest domain sizes that range from a few nanometers to many microns, depending strongly on the nature of the low-melting lipid. When POPC, which gives rise to nanodomains, is replaced by DOPC, which yields macrodomains, an intermediate region is observed of patterned, or modulated phases. This domain morphology can be explained as a competition between line tension and bending energies with patterns occurring when the two are nearly balanced. Necessary for testing this model are measurements of line tension, and bending moduli for both phases. Here we report the bending moduli of coexisting Lo and Ld phases from mixtures that produce domains ranging in size from nanoscopic to macroscopic. Measurements were made by shape analysis of giant unilamellar vesicles with both fluorescence and phase contrast microscopy. Vesicles of a single phase were made by the gentle hydration method to obtain a more narrow distribution of vesicle tensions than is obtained by electroformation. A transmembrane helical peptide, WALP23, strongly partitions to the liquid disordered phase, and changes the size of coexisting Lo + Ld domains. We are currently investigating the effects of WALP23 on membrane mechanical properties and line tension.

Studying the Membrane-Bound Conformation of Alpha-Synuclein using a Model Transmembrane Peptide System in a Lipid Bilayer

Alpha-synuclein is intrinsically disordered in solution but adopts a partially alpha-helical structure when membrane bound; we are currently investigating this structure using a cyanylated cysteine vibrational probe at various sites on the αS membrane binding domain. In order to quantitatively understand the depth dependence of the SCN probe in a lipid bilayer, four variants of a poly-L transmembrane helical peptide were designed and synthesized with cysteine residues at different depths in a membrane bilayer. A nonaqueous cyanylation and purification protocol was developed to introduce the probe group on these very hydrophobic model peptides. Vibrational spectroscopy was carried out on the four variants in a model lipid system using both solution/vesicle and oriented, immobilized samples. These data will help to elucidate the membrane interactions of both αS and other membrane-bound proteins.

Transmembrane-Peptide Structure Formation from Coarse-Grained Simulations

Interfacial systems are at the core of fascinating phenomena in many disciplines, such as biochemistry, soft-matter physics, and food science. However, the parametrization of accurate, reliable, and consistent coarse-grained (CG) models for systems at interfaces remains a challenging endeavor. In this work, we report recent advancements made toward the description of secondary-structure formation of peptides in a membrane environment using CG models. By combining a lipid model that can semi-quantitatively reproduce material properties of a fluid membrane bilayer [1] and a peptide model that is not biased toward one particular state (e.g., alpha-helix or beta-sheet) [2], the combined parametrization [3] allows to look at how peptide structure is affected by the membrane environment on long timescales. We illustrate the robustness of the model by folding different WALP transmembrane helical peptides starting from stretched, unstructured conformations using both simple canonical simulations and enhanced-sampling techniques [4]. Finally, the method is used to fold the 50-residue-long major pVIII coat protein (fd coat) of the filamentous fd bacteriophage. The results show excellent agreement with experimental structures and atomistic simulations in implicit membrane, demonstrating that such a protocol can serve as a starting point for better-refined atomistic simulations in a multiscale framework. [1] Wang and Deserno, J. Phys. Chem. B 114, 11207 (2010). [2] Bereau and Deserno, J. Chem. Phys. 130, 235106 (2009). [3] Bereau, Wang, and Deserno, J. Chem. Phys. 140, 115101 (2014). [4] Bereau and Deserno, submitted.

A designed buried salt bridge modulates heterodimerization of a membrane peptide.

Specific helix-helix interactions underpin the correct assembly of multipass membrane proteins. Here, we show that a designed buried salt bridge mediates heterodimer formation of model transmembrane helical peptides in a pH-dependent manner. The model peptides bear side chains functionalized with either a carboxylic acid or a primary amine within a hydrophobic segment. The association behavior was monitored by Förster resonance energy transfer, revealing that heterodimer formation is maximized at a pH close to neutrality (pH 6.5), at which each peptide is found in a charged state. In contrast, heterodimerization is disfavored at low and high values of pH, because either the carboxylic acid or the primary amine is present in its neutral state, thus preventing the formation of a salt bridge. These findings provide a blueprint for the design and modulation of protein-protein interactions in membrane proteins.

Enhanced Sampling of Coarse-Grained Transmembrane-Peptide Structure Formation from Hydrogen-Bond Replica Exchange

Protein structure formation in the membrane highlights a grand challenge of sampling in computer simulations, because kinetic traps and slow dynamics make it difficult to find the native state. Exploiting increased fluctuations at higher temperatures can help overcome free-energy barriers, provided the membrane's structure remains stable. In this work, we apply Hamiltonian replica-exchange molecular dynamics, where we only tune the backbone hydrogen-bond strength to help reduce the propensity of long-lived misfolded states. Using a recently developed coarse-grained model, we illustrate the robustness of the method by folding different WALP transmembrane helical peptides starting from stretched, unstructured conformations. We show the efficiency of the method by comparing to simulations without enhanced sampling, achieving folding in one example after significantly longer simulation times. Analysis of the bilayer structure during folding provides insight into the local membrane deformation during helix formation as a function of chain length (from 16 to 23 residues). Finally, we apply our method to fold the 50-residue-long major pVIII coat protein (fd coat) of the filamentous fd bacteriophage. Our results agree well with experimental structures and atomistic simulations based on implicit membrane models, suggesting that our explicit CG folding protocol can serve as a starting point for better-refined atomistic simulations in a multiscale framework.

Transferring the PRIMO Coarse-Grained Force Field to the Membrane Environment: Simulations of Membrane Proteins and Helix-Helix Association.

An extension of the recently developed PRIMO coarse-grained force field to membrane environments, PRIMO-M, is described. The membrane environment is modeled with the heterogeneous dielectric generalized Born (HDGB) methodology that simply replaces the standard generalized Born model in PRIMO without further parametrization. The resulting model was validated by comparing amino acid insertion free energy profiles and application in molecular dynamics simulations of membrane proteins and membrane-interacting peptides. Membrane proteins with 148–661 amino acids show stable root-mean-squared-deviations (RMSD) between 2 and 4 Å for most systems. Transmembrane helical peptides maintain helical shape and exhibit tilt angles in good agreement with experimental or other simulation data. The association of two glycophorin A (GpA) helices was simulated using replica exchange molecular dynamics simulations yielding the correct dimer structure with a crossing angle in agreement with previous studies. Finally, conformational sampling of the influenza fusion peptide also generates structures in agreement with previous studies. Overall, these findings suggest that PRIMO-M can be used to study membrane bound peptides and proteins and validates the transferable nature of the PRIMO coarse-grained force field.

PRIMO-M: An Extension of the Coarse-Grained Force Field Primo to the Membrane Environment

CG models for biomolecules and lipids are widely established but often suffer from a lack of transferability between different systems and to different environments. Furthermore, the combination with atomistic force fields in hybrid AA/CG multiscale models is often challenging. To alleviate these problems, we have previously developed the PRIMO coarse-grained force field. Here, the extension of PRIMO to membrane environments, PRIMO-M, is presented. The membrane environment is modeled implicitly with the heterogeneous dielectric generalized Born methodology that simply replaces the standard generalized Born model in PRIMO without further parameterization. The resulting model was initially validated by reproducing amino acid insertion free energy profiles. Membrane proteins with 148-661 amino acids show stable root-mean-squared-deviation between 2 and 4 A for most systems. PRIMO-M was able to predict tilt angles of several transmembrane helical peptides that are in good agreement with experimental or other simulation data. The association of two glycophorin A helices was simulated using replica exchange molecular dynamics simulations yielding the correct dimer structure with a crossing angle in agreement with previous studies. Finally, the conformational sampling of influenza fusion peptide also generates structures in agreement with previous studies. Overall, these findings suggest that PRIMO-M can be used to study membrane bound peptides and proteins and validates the transferable nature of the PRIMO coarse-grained force field.

Influence of a Central Tryptophan and of Cholesterol on the Properties of Defined Transmembrane Helical Peptides

GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide) provides a favorable host framework for investigations of the influence of guest amino acids, for example a third, centrally located, Trp residue, within the hydrophobic core of a well characterized transmembrane helix. It is crucial to note that the orientation and rotation of GWALP23 are sensitive to single-residue replacements, in part because the membrane-spanning helix exhibits only limited dynamic averaging of solid-state NMR observables such as the 2H quadrupolar splitting (Biophys. J. 101, 2939). We introduced a Trp residue into position 12 or 13 of GWALP23 (replacing either L12 or A13) and incorporated specific 2H-Ala labels within the helical core sequence. Solid-state 2H NMR spectra of GWALP23-W12 reveal that the peptide remains helical and retains a dominant preferred tilted transmembrane orientation with only a low extent of dynamic averaging, comparable to GWALP23 itself. Indeed, the tilt and dynamics of GWALP23-W12 are quite similar to the values observed for GWALP23 in DMPC bilayer membranes. We have analyzed the dynamics of the peptide helices using a modified Gaussian treatment as well as a semi-static treatment. The results indicate that a central Trp residue at position 12 does not appreciably perturb the properties of bilayer-spanning GWALP23. (By contrast, Arg-12 or Lys-12, when charged, induces multi-state behavior for GWALP23 in bilayer membranes [PNAS 110, 1692].) Additionally, we are investigating the influence of cholesterol upon the properties of membrane-spanning GWALP23, GWALP23-W12 and GWALP23-K12.

Characterizing Moderately Short Antimicrobial Tryptophan/Arginine-Rich Peptides

Antimicrobial peptides offer attractive alternatives to the problem of increasing bacterial drug resistance. To address this issue, we have engineered moderately short “hybrid” peptides that are somewhat longer than the antimicrobial surface-acting lactoferrin-derived LfB6 (RRWQWR-NH2), yet shorter than the inert model transmembrane helical peptide RWALP23 (acetyl-GRALW(LA)6LWLARA-NH2). Previously we observed that a 15-residue peptide with 4 arginines, acetyl-GRRW(LA)3LWRRA-NH2 (designated RRWALP15) exhibits significant activity against E. coli and S. aureus (MIC ∼10 μg/ml). To further interrogate the importance of peptide length and detailed amino acid sequence, we have synthesized additional LfB-RWALP hybrid peptides that have between 9 and 15 total residues and differing numbers of arginine and tryptophan residues. Into peptides such as, acetyl-GRRWWAWWRRA-NH2, acetyl-GRRWLALWRRA-NH2, and acetyl-GRWLALALALWRA-NH2, we incorporate 2H-labeled alanine residues and seek to compare the biophysical properties and the antimicrobial potencies of several such peptides. Solid-state deuterium (2H) NMR spectra suggest peptide conformational heterogeneity in several different mechanically aligned lipid bilayers of varying lipid thickness. The circular dichroism spectra of the new LfB-KWALP hybrid peptides in lipid vesicles furthermore reveal, in most cases, a mixture α-helical and β-like conformations. The antimicrobial activity typically is higher when four arginines are present, yet the activity does not appear to correlate directly with folding or secondary structure.

Gadolinium(III) Spin Labels for High‐Sensitivity Distance Measurements in Transmembrane Helices

Distance determination, by pulse EPR techniques, between two spin labels attached to biomolecules has become an attractive methodology to probe conformations and assemblies of biomolecules in frozen solutions. Among these techniques, double electron-electron resonance (DEER or PELDOR), which can access distances in the range of 1.7 to 8 nm, is highly popular, and the most widely used spin labels are nitroxide radicals. Membrane proteins in their natural environment are of particular interest for DEER applications, since those pose a considerable challenge for Xray crystallography and NMR spectroscopy. DEER studies of peptides and proteins in either reconstituted or model membranes are considerably more challenging than those in solution, because the high local concentration of the spins in the membrane decreases the phase memory time and, therefore, sensitivity. Most DEER measurements on nitroxide-labeled biomolecules are carried out at X-band frequencies (9.5 GHz, 0.35 T), and recently such measurements were demonstrated in frozen cells. A major difficulty of such measurements is the reduction of nitroxides in the cell, which severely limits the scope of such exciting developments. Recently, Gd (S= 7/2) spin labels have been suggested as an alternative to nitroxide spin labels for W-band and Qband DEER distance measurements. Gd tags can be attached to proteins, similar to nitroxides, by site-directed spin labeling (SDSL). Gd features high sensitivity at high frequencies and the DEER measurements are free of orientation selection effects so that the distance distribution can readily be extracted from a single DEER measurement. Moreover, in the context of future development of in-cell DEER measurements, Gd chelates are stable under in vivo conditions as known from their applications as contrast agents for magnetic resonance imaging (MRI). Gd-Gd DEER has been demonstrated on model systems, proteins, peptides, and DNA, all in isotropic membrane-free solutions. Distance measurements between a Gd label and a nitroxide label have also been shown to yield attractive sensitivity. In this work, we continue to develop the approach of Gd–Gd DEER distance measurements and demonstrate for the first time such measurements in a model membrane. The model system we chose consists of the well-studied transmembrane helical WALP peptides in 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC) vesicles. We demonstrate the sensitivity of W-band Gd–Gd DEER to small distance variations in a membrane. Using WALP peptides of different lengths we show that such measurements pick up, in addition to the helix extension, also subtle “cis–trans” effects arising from different positions of the labels with respect to the helix axes. In addition, we report the effect of the spin label interaction with the membranes on the measured distance distribution. We compared W-band DEER on WALP23 labeled with two different Gd tags with X-band DEER onWALP23 labeled with nitroxide tags. Here we used X-band, rather than W-band, to avoid complications owing to orientation selection. The differences observed are important and suggest that by employing different spin labels such effects can be isolated. Finally, we show that the effect of hydrophobic mismatch between peptide and membrane can be explored by Gd–Gd DEER. WALP23 was labeled at the N and C termini (see Table 1) with two nitroxides (WAL23-NO) using (l-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)methanesulfonate (MTSSL) and two different Gd-DOTA derivatives, shown in Figure 1. WALP23-DOTA is labeled with a DOTA chelate and WALP23-C1 with DOTA with phenylethylamine substituents. The bulky substituents were designed to restrict the flexibility of the tag. The hydrophobic length of WALP23 (ca. 2.6 nm) is very close to that of the hydrophobic thickness of DOPC bilayers (ca. 2.7 nm). The sample composition was 50 mm WALP23 in DOPC multilamellar vesicles (MLV; 1:1000 peptide/lipid molar ratio). Details of the sample preparation are given in the Supporting Information. [*] Dr. E. Matalon, Dr. A. Feintuch, Prof. D. Goldfarb Department of Chemical Physics, Weizmann Institute of Science Rehovot, 76100 (Israel) E-mail: Daniella.goldfarb@weizmann.ac.il