Spiropyran-Based Photoisomerizable α-Amino Acid for Membrane-Active Peptide Modification.

Photoisomerizable peptides are promising drug candidates in photopharmacology. While azobenzene- and diarylethene-containing photoisomerizable peptides have already demonstrated their potential in this regard, reports on the use of spiropyrans to photoregulate bioactive peptides are still scarce. This work focuses on the design and synthesis of a spiropyran-derived amino acid, (S)-2-amino-3-(6'-methoxy-1',3',3'-trimethylspiro-[2H-1-benzopyran-2,2'-indolin-6-yl])propanoic acid, which is suitable for the preparation of photoisomerizable peptides. The utility of this amino acid is demonstrated by incorporating it into the backbone of BP100, a known membrane-active peptide, and by examining the photoregulation of the membrane perturbation by the spiropyran-containing peptides. The toxicity of the peptides (against the plant cell line BY-2), their bacteriotoxicity (E. coli), and actin-auxin oscillator modulation ability were shown to be significantly dependent on the photoisomeric state of the spiropyran unit.

Length matters: Functional flip of the short TatA transmembrane helix.

The twin arginine translocase (Tat) exports folded proteins across bacterial membranes. The putative pore-forming or membrane-weakening component (TatAd in B. subtilis) is anchored to the lipid bilayer via an unusually short transmembrane α-helix (TMH), with less than 16 residues. Its tilt angle in different membranes was analyzed under hydrophobic mismatch conditions, using synchrotron radiation circular dichroism and solid-state NMR. Positive mismatch (introduced either by reconstitution in short-chain lipids or by extending the hydrophobic TMH length) increased the helix tilt of the TMH as expected. Negative mismatch (introduced either by reconstitution in long-chain lipids or by shortening the TMH), on the other hand, led to protein aggregation. These data suggest that the TMH of TatA is just about long enough for stable membrane insertion. At the same time, its short length is a crucial factor for successful translocation, as demonstrated here in native membrane vesicles using an in vitro translocation assay. Furthermore, when reconstituted in model membranes with negative spontaneous curvature, the TMH was found to be aligned parallel to the membrane surface. This intrinsic ability of TatA to flip out of the membrane core thus seems to play a key role in its membrane-destabilizing effect during Tat-dependent translocation.

Highly Fluorinated Peptide Probes with Enhanced In Vivo Stability for 19 F-MRI.

A labeling strategy for in vivo 19 F-MRI (magnetic resonance imaging) based on highly fluorinated, short hydrophilic peptide probes, is developed. As dual-purpose probes, they are functionalized further by a fluorophore and an alkyne moiety for bioconjugation. High fluorination is achieved by three perfluoro-tert-butyl groups, introduced into asparagine analogues by chemically stable amide bond linkages. d-amino acids and ß-alanine in the sequences endow the peptide probes with low cytotoxicity and high serum stability. This design also yielded unstructured peptides, rendering all 27 19 F substitutions chemically equivalent, giving rise to a single 19 F-NMR resonance with <10 Hz linewidth. The resulting performance in 19 F-MRI is demonstrated for six different peptide probes. Using fluorescence microscopy, these probes are found to exhibit high stability and long circulation times in living zebrafish embryos. Furthermore, the probes can be conjugated to bovine serum albumin with only amoderate increase in 19 F-NMR linewidth to ≈30 Hz. Overall, these peptide probes are hence suitable for in vivo 19 F-MRI applications.

Probing and Manipulating the Lateral Pressure Profile in Lipid Bilayers Using Membrane-Active Peptides-A Solid-State 19F NMR Study.

The lateral pressure profile constitutes an important physical property of lipid bilayers, influencing the binding, insertion, and function of membrane-active peptides, such as antimicrobial peptides. In this study, we demonstrate that the lateral pressure profile can be manipulated using the peptides residing in different regions of the bilayer. A 19F-labeled analogue of the amphiphilic peptide PGLa was used to probe the lateral pressure at different depths in the membrane. To evaluate the lateral pressure profile, we measured the orientation of this helical peptide with respect to the membrane using solid-state 19F-NMR, which is indicative of its degree of insertion into the bilayer. Using this experimental approach, we observed that the depth of insertion of the probe peptide changed in the presence of additional peptides and, furthermore, correlated with their location in the membrane. In this way, we obtained a tool to manipulate, as well as to probe, the lateral pressure profile in membranes.

Tough, Transparent, 3D-Printable, and Self-Healing Poly(ethylene glycol)-Gel (PEGgel).

Polymer gels, such as hydrogels, have been widely used in biomedical applications, flexible electronics, and soft machines. Polymer network design and its contribution to the performance of gels has been extensively studied. In this study, the critical influence of the solvent nature on the mechanical properties and performance of soft polymer gels is demonstrated. A polymer gel platform based on poly(ethylene glycol) (PEG) as solvent is reported (PEGgel). Compared to the corresponding hydrogel or ethylene glycol gel, the PEGgel with physically cross-linked poly(hydroxyethyl methacrylate-co-acrylic acid) demonstrates high stretchability and toughness, rapid self-healing, and long-term stability. Depending on the molecular weight and fraction of PEG, the tensile strength of the PEGgels varies from 0.22 to 41.3 MPa, fracture strain from 12% to 4336%, modulus from 0.08 to 352 MPa, and toughness from 2.89 to 56.23 MJ m-3 . Finally, rapid self-healing of the PEGgel is demonstrated and a self-healing pneumatic actuator is fabricated by 3D-printing. The enhanced mechanical properties of the PEGgel system may be extended to other polymer networks (both chemically and physically cross-linked). Such a simple 3D-printable, self-healing, and tough soft material holds promise for broad applications in wearable electronics, soft actuators and robotics.

Membrane-Mediated Activity of Local Anesthetics.

The activity of local anesthetics (LAs) has been attributed to the inhibition of ion channels, causing anesthesia. However, there is a growing body of research showing that LAs act on a wide range of receptors and channel proteins far beyond simple analgesia. The current concept of ligand recognition may no longer explain the multitude of protein targets influenced by LAs. We hypothesize that LAs can cause anesthesia without directly binding to the receptor proteins just by changing the physical properties of the lipid bilayer surrounding these proteins and ion channels based on LAs' amphiphilicity. It is possible that LAs act in one of the following ways: They 1) dissolve raft-like membrane microdomains, 2) impede nerve impulse propagation by lowering the lipid phase transition temperature, or 3) modulate the lateral pressure profile of the lipid bilayer. This could also explain the numerous additional effects of LAs besides anesthesia. Furthermore, the concepts of membrane-mediated activity and binding to ion channels do not have to exclude each other. If we were to consider LA as the middle part of a continuum between unspecific membrane-mediated activity on one end and highly specific ligand binding on the other end, we could describe LA as the link between the unspecific action of general anesthetics and toxins with their highly specific receptor binding. This comprehensive membrane-mediated model offers a fresh perspective to clinical and pharmaceutical research and therapeutic applications of local anesthetics. SIGNIFICANCE STATEMENT: Local anesthetics, according to the World Health Organization, belong to the most important drugs available to mankind. Their rediscovery as therapeutics and not only anesthetics marks a milestone in global pain therapy. The membrane-mediated mechanism of action proposed in this review can explain their puzzling variety of target proteins and their thus far inexplicable therapeutic effects. The new concept presented here places LAs on a continuum of structures and molecular mechanisms in between small general anesthetics and the more complex molecular toxins.

Structural and functional characterization of the pore-forming domain of pinholin S2168.

Pinholin S2168 triggers the lytic cycle of bacteriophage φ21 in infected Escherichia coli Activated transmembrane dimers oligomerize into small holes and uncouple the proton gradient. Transmembrane domain 1 (TMD1) regulates this activity, while TMD2 is postulated to form the actual "pinholes." Focusing on the TMD2 fragment, we used synchrotron radiation-based circular dichroism to confirm its α-helical conformation and transmembrane alignment. Solid-state 15N-NMR in oriented DMPC bilayers yielded a helix tilt angle of τ = 14°, a high order parameter (Smol = 0.9), and revealed the azimuthal angle. The resulting rotational orientation places an extended glycine zipper motif (G40xxxS44xxxG48) together with a patch of H-bonding residues (T51, T54, N55) sideways along TMD2, available for helix-helix interactions. Using fluorescence vesicle leakage assays, we demonstrate that TMD2 forms stable holes with an estimated diameter of 2 nm, as long as the glycine zipper motif remains intact. Based on our experimental data, we suggest structural models for the oligomeric pinhole (right-handed heptameric TMD2 bundle), for the active dimer (right-handed Gly-zipped TMD2/TMD2 dimer), and for the full-length pinholin protein before being triggered (Gly-zipped TMD2/TMD1-TMD1/TMD2 dimer in a line).

Shape-Memory Effect by Sequential Coupling of Functions over Different Length Scales in an Architectured Hydrogel.

The integration of functions in materials in order to gain macroscopic effects in response to environmental changes is an ongoing challenge in material science. Here, functions on different hierarchical levels are sequentially linked to translate a pH-triggered conformational transition from the molecular to the macroscopic level to induce directed movements in hydrogels. When the pH is increased, lysine-rich peptide molecules change their conformation into a ß-hairpin structure because of the reduced electrostatic repulsion among the deprotonated amino groups. Coupled to this conformation change is the capability of the ß-hairpin motifs to subsequently assemble into aggregates acting as reversible cross-links, which are used as controlling units to fix a temporary macroscopic shape. A structural function implemented into the hydrogel by a microporous architecture-enabled nondisruptive deformation upon compression by buckling of pore walls and their elastic recovery. Coupled to this structural function is the capability of the porous material to enhance the diffusion of ions into the hydrogel and to keep the dimension of the macroscopic systems almost constant when the additional cross-links are formed or cleaved as it limits the dimensional change of the pore walls. Covalent cross-linking of the hydrogel into a polymer network acted as gear shift to ensure translation of the function on the molecular level to the macroscopic dimension. In this way, the information of a directed shape-shift can be programmed into the material by mechanical deformation and pH-dependent formation of temporary net points. The information could be read out by lowering the pH. The peptides reverted back into their original random coil conformation and the porous polymer network could recover from the previously applied elastic deformation. The level of multifunctionality of the hydrogels can be increased by implementation of additional orthogonal functions such as antimicrobicity by proper selection of multifunctional peptides, which could enable sophisticated biomedical devices.

Monofluoroalkene-Isostere as a 19 F†NMR Label for the Peptide Backbone: Synthesis and Evaluation in Membrane-Bound PGLa and (KIGAKI)3.

Solid-state 19 F NMR is a powerful method to study the interactions of biologically active peptides with membranes. So far, in labelled peptides, the 19 F-reporter group has always been installed on the side chain of an amino acid. Given the fact that monofluoroalkenes are non-hydrolyzable peptide bond mimics, we have synthesized a monofluoroalkene-based dipeptide isostere, Val-Ψ[(Z)-CF=CH]-Gly, and inserted it in the sequence of two well-studied antimicrobial peptides: PGLa and (KIGAKI)3 are representatives of an α-helix and a ß-sheet. The conformations and biological activities of these labeled peptides were studied to assess the suitability of monofluoroalkenes for 19 F NMR structure analysis.

Bilayer thickness determines the alignment of model polyproline helices in lipid membranes.

Our understanding of protein folds relies fundamentally on the set of secondary structures found in the proteomes. Yet, there also exist intriguing structures and motifs that are underrepresented in natural biopolymeric systems. One example is the polyproline II helix, which is usually considered to have a polar character and therefore does not form membrane spanning sections of membrane proteins. In our work, we have introduced specially designed polyproline II helices into the hydrophobic membrane milieu and used 19F NMR to monitor the helix alignment in oriented lipid bilayers. Our results show that these artificial hydrophobic peptides can adopt several different alignment states. If the helix is shorter than the thickness of the hydrophobic core of the membrane, it is submerged into the bilayer with its long axis parallel to the membrane plane. The polyproline helix adopts a transmembrane alignment when its length exceeds the bilayer thickness. If the peptide length roughly matches the lipid thickness, a coexistence of both states is observed. We thus show that the lipid thickness plays a determining role in the occurrence of a transmembrane polyproline II helix. We also found that the adaptation of polyproline II helices to hydrophobic mismatch is in some notable aspects different from α-helices. Finally, our results prove that the polyproline II helix is a competent structure for the construction of transmembrane peptide segments, despite the fact that no such motif has ever been reported in natural systems.

Biocompatibility of Amine-Functionalized Silica Nanoparticles: The Role of Surface Coverage.

Here, amorphous silica nanoparticles (NPs), one of the most abundant nanomaterials, are used as an example to illustrate the utmost importance of surface coverage by functional groups which critically determines biocompatibility. Silica NPs are functionalized with increasing amounts of amino groups, and the number of surface exposed groups is quantified and characterized by detailed NMR and fluorescamine binding studies. Subsequent biocompatibility studies in the absence of serum demonstrate that, irrespective of surface modification, both plain and amine-modified silica NPs trigger cell death in RAW 264.7 macrophages. The in vitro results can be confirmed in vivo and are predictive for the inflammatory potential in murine lungs. In the presence of serum proteins, on the other hand, a replacement of only 10% of surface-active silanol groups by amines is sufficient to suppress cytotoxicity, emphasizing the relevance of exposure conditions. Mechanistic investigations identify a key role of lysosomal injury for cytotoxicity only in the presence, but not in the absence, of serum proteins. In conclusion, this work shows the critical need to rigorously characterize the surface coverage of NPs by their constituent functional groups, as well as the impact of serum, to reliably establish quantitative nanostructure activity relationships and develop safe nanomaterials.

Transmembrane Polyproline Helix.

The third most abundant polypeptide conformation in nature, the polyproline-II helix, is a polar, extended secondary structure with a local organization stabilized by intercarbonyl interactions within the peptide chain. Here we design a hydrophobic polyproline-II helical peptide based on an oligomeric octahydroindole-2-carboxylic acid scaffold and demonstrate its transmembrane alignment in model lipid bilayers by means of solid-state 19F NMR. As result, we provide a first example of a purely artificial transmembrane peptide with a structural organization that is not based on hydrogen-bonding.

Orthogonal 19 F-Labeling for Solid-State NMR Spectroscopy Reveals the Conformation and Orientation of Short Peptaibols in Membranes.

Peptaibols are promising drug candidates in view of their interference with cellular membranes. Knowledge of their lipid interactions and membrane-bound structure is needed to understand their activity and should be, in principle, accessible by solid-state NMR spectroscopy. However, their unusual amino acid composition and noncanonical conformations make it very challenging to find suitable labels for NMR spectroscopy. Particularly in the case of short sequences, new strategies are required to maximize the structural information that can be obtained from each label. Herein, l-3-(trifluoromethyl)bicyclopent[1.1.1]-1-ylglycine, (R)- and (S)-trifluoromethylalanine, and 15 N-backbone labels, each probing a different direction in the molecule, have been combined to elucidate the conformation and membrane alignment of harzianin HK-VI. For the short sequence of 11 amino acids, 12 orientational constraints have been obtained by using 19 F and 15 N NMR spectroscopy. This strategy revealed a ß-bend ribbon structure, which becomes realigned in the membrane from a surface-parallel state towards a membrane-spanning state, with increasing positive spontaneous curvature of the lipids.

"Force-from-lipids" gating of mechanosensitive channels modulated by PUFAs.

The level of fatty acid saturation in phospholipids is a crucial determinant of the biophysical properties of the lipid bilayer. Integral membrane proteins are sensitive to changes of their bilayer environment such that their activities and localization can be profoundly affected. When incorporated into phospholipids of mammalian cells, poly-unsaturated fatty acids (PUFAs) determine the mechanical properties of the bilayer thereby affecting several membrane-associated functions such as endo- and exo-cytosis and ion channel/membrane receptor signalling cascades. In order to understand how membrane tension is propagated through poly-unsaturated bilayers, we characterized the effect of lipid saturation on liposome reconstituted MscS and MscL, the two bacterial mechanosensitive ion channels that have for many years served as models of ion- channel-mediated mechanotransduction. The combination of NMR and patch clamp experiments in this study demonstrate that bilayer thinning is the main responsible factor for the modulation of the MscL threshold of activation while a change in transbilayer pressure profile is indicated as the main factor behind the observed modulation of the MscS kinetics. Together, our data offer a novel insight into how the structural shape differences between the two types of mechanosensitive channels determine their differential modulation by poly-unsaturated phospholipids and thus lay the foundation for future functional studies of eukaryotic ion channels involved in the physiology of mechanosensory transduction processes in mammalian cells. SUMMARY: Mechanosensitive channels MscL and MscS are differentially modulated by poly-unsaturated fatty acids in lipid bilayers. MscL becomes sensitized because of increased hydrophobic mismatch while MscS open state is stabilized due to changes in the bilayer lateral pressure profile determined by NMR.

Structural Behavior of the Peptaibol Harzianin HK VI in a DMPC Bilayer: Insights from MD Simulations.

Microsecond molecular dynamics simulations of harzianin HK VI (HZ) interacting with a dimyristoylphosphatidylcholine bilayer were performed at the condition of low peptide-to-lipid ratio. Two orientations of HZ molecule in the bilayer were found and characterized. In the orientation perpendicular to the bilayer surface, HZ induces a local thinning of the bilayer. When inserted into the bilayer parallel to its surface, HZ is located nearly completely within the hydrophobic region of the bilayer. A combination of solid-state NMR and circular dichroism experiments found the latter orientation to be dominant. An extended sampling simulation provided qualitative results and showed the same orientation to be a global minimum of free energy. The secondary structure of HZ was characterized, and it was found to be located in the 310-helical family. The specific challenges of computer simulation of nonpolar peptides are discussed briefly.

Orientation and Location of the Cyclotide Kalata B1 in Lipid Bilayers Revealed by Solid-State NMR.

Cyclotides are ultra-stable cyclic disulfide-rich peptides from plants. Their biophysical effects and medically interesting activities are related to their membrane-binding properties, with particularly high affinity for phosphatidylethanolamine lipids. In this study we were interested in understanding the molecular details of cyclotide-membrane interactions, specifically with regard to the spatial orientation of the cyclotide kalata B1 from Oldenlandia affinis when embedded in a lipid bilayer. Our experimental approach was based on the use of solid-state 19F-NMR of oriented bilayers in conjunction with the conformationally restricted amino acid L-3-(trifluoromethyl)bicyclopent-[1.1.1]-1-ylglycine as an orientation-sensitive 19F-NMR probe. Its rigid connection to the kalata B1 backbone scaffold, together with the well-defined structure of the cyclotide, allowed us to calculate the protein alignment in the membrane directly from the orientation-sensitive 19F-NMR signal. The hydrophobic and polar residues on the surface of kalata B1 form well-separated patches, endowing this cyclotide with a pronounced amphipathicity. The peptide orientation, as determined by NMR, showed that this amphipathic structure matches the polar/apolar interface of the lipid bilayer very well. A location in the amphiphilic headgroup region of the bilayer was supported by 15N-NMR of uniformly labeled protein, and confirmed using solid-state 31P- and 2H-NMR. 31P-NMR relaxation data indicated a change in lipid headgroup dynamics induced by kalata B1. Changes in the 2H-NMR order parameter profile of the acyl chains suggest membrane thinning, as typically observed for amphiphilic peptides embedded near the polar/apolar bilayer interface. Furthermore, from the 19F-NMR analysis two important charged residues, E7 and R28, were found to be positioned equatorially. The observed location thus would be favorable for the postulated binding of E7 to phosphatidylethanolamine lipid headgroups. Furthermore, it may be speculated that this pair of side chains could promote oligomerization of kalata B1 through electrostatic intermolecular contacts via their complementary charges.

Room-Temperature High-Efficiency Solid-State Triplet-Triplet Annihilation Up-Conversion in Amorphous Poly(olefin sulfone)s.

Triplet-triplet annihilation up-conversion (TTA-UC) is a developing technology that can enable spectral conversion under sunlight. Previously, it was found that efficient TTA-UC can be realized in polymer hosts for temperatures above the polymer's glass transition (T > Tg). In contrast, TTA-UC with high quantum yield for temperatures below Tg is rarely reported. In this article, we report new polymer hosts in which efficient TTA-UC is observed well below Tg, when the polymer is in a fully solid state. The four poly(olefin sulfone) hosts were loaded with upconversion dyes, and absolute quantum yields of TTA-UC (ηTTA-UC) were measured. The highest value of ηTTA-UC = 2.1% was measured for poly(1-dodecene sulfone). Importantly, this value was the same in vacuum and at ambient conditions, indicating that the host material acts as a good oxygen barrier. We performed time-resolved luminescence experiments in order to elucidate the impact of elementary steps of TTA-UC. In addition to optical characterization, we used magic angle spinning solid-state NMR experiments to estimate the T2 transverse relaxation time. Relatively long T2 times measured for poly(olefin sulfone)s indicate an enhanced nanoscale fluidity in the studied (co)polymers, which unexpectedly coexists with a rigidity on the macroscale. This would explain the exceptional triplet energy transfer between the guest molecules, despite the macroscopic rigidity.

Design, Synthesis, and Application of an Optimized Monofluorinated Aliphatic Label for Peptide Studies by Solid-State 19 F†NMR Spectroscopy.

A conformationally restricted monofluorinated α-amino acid, (3-fluorobicyclo[1.1.1]pentyl)glycine (F-Bpg), was designed as a label for the structural analysis of membrane-bound peptides by solid-state 19 F NMR spectroscopy. The compound was synthesized and validated as a 19 F label for replacing natural aliphatic α-amino acids. Calculations suggested that F-Bpg is similar to Leu/Ile in terms of size and lipophilicity. The 19 F NMR label was incorporated into the membrane-active antimicrobial peptide PGLa and provided information on the structure of the peptide in a lipid bilayer.

Membrane Thinning and Thickening Induced by Membrane-Active Amphipathic Peptides.

Membrane thinning has been discussed as a fundamental mechanism by which antimicrobial peptides can perturb cellular membranes. To understand which factors play a role in this process, we compared several amphipathic peptides with different structures, sizes and functions in their influence on the lipid bilayer thickness. PGLa and magainin 2 from X. laevis were studied as typical representatives of antimicrobial cationic amphipathic α-helices. A 1:1 mixture of these peptides, which is known to possess synergistically enhanced activity, allowed us to evaluate whether and how this synergistic interaction correlates with changes in membrane thickness. Other systems investigated here include the α-helical stress-response peptide TisB from E. coli (which forms membrane-spanning dimers), as well as gramicidin S from A. migulanus (a natural antibiotic), and BP100 (designer-made antimicrobial and cell penetrating peptide). The latter two are very short, with a circular ß-pleated and a compact α-helical structure, respectively. Solid-state (2)H-NMR and grazing incidence small angle X-ray scattering (GISAXS) on oriented phospholipid bilayers were used as complementary techniques to access the hydrophobic thickness as well as the bilayer-bilayer repeat distance including the water layer in between. This way, we found that magainin 2, gramicidin S, and BP100 induced membrane thinning, as expected for amphiphilic peptides residing in the polar/apolar interface of the bilayer. PGLa, on the other hand, decreased the hydrophobic thickness only at very high peptide:lipid ratios, and did not change the bilayer-bilayer repeat distance. TisB even caused an increase in the hydrophobic thickness and repeat distance. When reconstituted as a mixture, PGLa and magainin 2 showed a moderate thinning effect which was less than that of magainin 2 alone, hence their synergistically enhanced activity does not seem to correlate with a modulation of membrane thickness. Overall, the absence of a typical thinning response in the case of PGLa, and the increase in the repeat distance and membrane thickening observed for TisB, demonstrate that the concept of peptide-induced membrane thinning cannot be generalized. Instead, these results suggest that different factors contribute to the resulting changes in membrane thickness, such as the peptide orientation in the bilayer, and/or bilayer adaptation to hydrophobic mismatch.

Hydrophobic Mismatch Drives the Interaction of E5 with the Transmembrane Segment of PDGF Receptor.

The oncogenic E5 protein from bovine papillomavirus is a short (44 amino acids long) integral membrane protein that forms homodimers. It activates platelet-derived growth factor receptor (PDGFR) ß in a ligand-independent manner by transmembrane helix-helix interactions. The nature of this recognition event remains elusive, as numerous mutations are tolerated in the E5 transmembrane segment, with the exception of one hydrogen-bonding residue. Here, we examined the conformation, stability, and alignment of the E5 protein in fluid lipid membranes of substantially varying bilayer thickness, in both the absence and presence of the PDGFR transmembrane segment. Quantitative synchrotron radiation circular dichroism analysis revealed a very long transmembrane helix for E5 of ∼26 amino acids. Oriented circular dichroism and solid-state (15)N-NMR showed that the alignment and stability of this unusually long segment depend critically on the membrane thickness. When reconstituted alone in exceptionally thick DNPC lipid bilayers, the E5 helix was found to be inserted almost upright. In moderately thick bilayers (DErPC and DEiPC), it started to tilt and became slightly deformed, and finally it became aggregated in conventional DOPC, POPC, and DMPC membranes due to hydrophobic mismatch. On the other hand, when E5 was co-reconstituted with the transmembrane segment of PDGFR, it was able to tolerate even the most pronounced mismatch and was stabilized by binding to the receptor, which has the same hydrophobic length. As E5 is known to activate PDGFR within the thin membranes of the Golgi compartment, we suggest that the intrinsic hydrophobic mismatch of these two interaction partners drives them together. They seem to recognize each other by forming a closely packed bundle of mutually aligned transmembrane helices, which is further stabilized by a specific pair of hydrogen-bonding residues.