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The twin arginine translocase (Tat) exports folded proteins across bacterial membranes. The putative pore-forming or membrane-weakening component (TatA

A labeling strategy for in vivo

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.

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).

Fluorographene (FG) ultra-thin films are directly grown on flexible and non-flexible insulating silicate substrates and are characterized in terms of their structural and electronic properties. FG ...

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 β-sheet. The conformations and biological activities of these labeled peptides were studied to assess the suitability of monofluoroalkenes for 19 F NMR structure analysis.

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.

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.

In this chapter we give an overview of the use of 19F NMR for the study of biomembranes. Due to its unique sensitivity and small size, fluorine may be considered to bridge the gap between NMR on the one hand, and ESR and fluorescence on the other hand. Conventional NMR isotope labels do not alter the studied molecules but possess only low sensitivity, while ESR and fluorescence labels provide higher sensitivity but also represent a larger modification of the system of interest. The high sensitivity to its environment, large anisotropies and strong dipolar couplings render 19 F a useful NMR label to probe structure and dynamics in biomembranes, and to study intermolecular interactions. Fluorine has been introduced into numerous constituents of biomembranes, such as lipids, sterols as well as membrane associated peptides and integral membrane proteins. In particular, a growing set of fluorine-labeled amino acids has allowed one to address selected structural information in membrane proteins and peptides. The large anisotropy of 19F NMR interactions in the solid-state has been used to measure molecular orientations with respect to the membrane normal, allowing characterization, for example, of the function of membrane-active peptides. Distance measurements exploring the large distance range of 19F have been beneficial in the study of intermolecular interactions, to reveal oligomeric architectures of membrane proteins and/or transmembrane peptides. The sensitivity of the 19F chemical shift to the environment makes it possible to probe the molecular environment and detect even solvent isotope effects, this way revealing conformational changes and solvent/lipid accessibilities. Given the constraints in placing fluorine into biomembranes and embedded molecules, 19F NMR may not be a universal technique, but it certainly provides a powerful tool to answer specific questions regarding the structure and dynamics in biomembranes that would be difficult to solve otherwise.

Pinholin S(21)68 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 (15)N-NMR in oriented DMPC bilayers yielded a helix tilt angle of τ = 14°, a high order parameter (S(mol) = 0.9), and revealed the azimuthal angle. The resulting rotational orientation places an extended glycine zipper motif (G(40)xxxS(44)xxxG(48)) together with a patch of H-bonding residues (T(51), T(54), N(55)) 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).

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 β-hairpin structure because of the reduced electrostatic repulsion among the deprotonated amino groups. Coupled to this conformation change is the capability of the β-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.

Polymer gels, such as hydrogels, consisting of cross-linked polymer chains and small molecule solvents, have been widely used in biomedical applications, flexible electronics and soft machines due to the similarity of their mechanical properties to those of soft biological tissues. Polymer network design and its contribution to the properties of such materials has been extensively studied. In this study, we demonstrate the critical influence of the solvent nature on the mechanical properties and performance of soft polymer gels. Here, we report a polymer gel system (PEGgel) based on a poly(hydroxyethyl methacrylate-co-acrylic acid) copolymerized in the presence of poly(ethylene glycol) (PEG) used as the liquid phase of the PEGgel. Compared to the corresponding hydrogel or ethylene glycol based gel, the PEGgel demonstrates exceptional physical properties, such as high stretchability and toughness, rapid self-healing, and long-term stability under ambient conditions. Depending on the molecular weight and fraction of PEG, the tensile strength of PEGgels varied from 0.22 MPa to 41.3 MPa, fracture strain from 12% to 4336%, modulus from 0.08 MPa to 352 MPa, and toughness from 2.89 MJ m-3 to 56.23 MJ m-3 . The influence of PEG on the mechanical properties was evaluated using the coarse-grain molecular dynamics (CGMD) model and solid-state NMR, revealing abundant weak hydrogen bonding leading to the observed enhancement of mechanical properties. Finally, we demonstrated rapid self-healing of PEGgel materials and fabricated a self-healing pneumatic actuator by 3D printing of PEGgel structures. The enhanced mechanical properties of the PEGgel system could be extended to other polymer networks (both chemically and physically cross-linked). This study demonstrates the important influence of the liquid phase of polymer gels on their mechanical and other properties, and highlights the potential for finetuning different physical properties of gels through the use of PEG possessing weak but multiple hydrogen bond interactions with the polymer network. Such a simple 3D printable, self-healing and tough soft material holds promise for broad applications in wearable electronics, soft actuators and robotics. This article is protected by copyright. All rights reserved.

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.

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 β-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.

Low Z element based room temperature ferromagnetic materials are highly sought after for many applications. In this work, we report the development of fluorine functionalized graphene-hBN out-of-plane heterostructures (hBNCF), which possess large room temperature ferromagnetic ordering (magnetization ∼ 0.05 emu/g and coercivity ∼ 420 Oe) and exhibit T2 relaxation enhancement (0.55 s−1 found for D2O was increased to ∼7 s−1 at 1 mg/ml) in water without causing the T1. The ferromagnetic nature of the hBNCF powder devoid of any metallic magnetic impurities was further shown by magnetic force microscopy. Layered structures of hBNCF that have an optical bandgap of ∼3.89 eV were produced using a two step method: Shear exfoliated hBN layers were modified by surface graphitisation to result into layered graphene heterostructures (hBNC), which were then functionalized with fluorine. Apparent ferromagnetic properties arise from these large scale, vertical, non-van der Waals connected, crystalline layer structures. Density functional calculations show the role of out-of-plane covalently connected BN domains and their interaction with fluorine, that causes the formation of new states in the graphitic lattice. These states exhibit a spin splitting, which contributes to the observed large magnetic ordering.

Solid-state

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.

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.

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) β 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 15N-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.

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.

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

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.

Free to read at publisher's site. 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.

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 ...

In this work, we present experimental data, supported by a quantitative model, on the generation and effect of potential gradients across a tethered bilayer lipid membrane (tBLM) with, to the best of our knowledge, novel architecture. A challenge to generating potential gradients across tBLMs arises from the tethering coordination chemistry requiring an inert metal such as gold, resulting in any externally applied voltage source being capacitively coupled to the tBLM. This in turn causes any potential across the tBLM assembly to decay to zero in milliseconds to seconds, depending on the level of membrane conductance. Transient voltages applied to tBLMs by pulsed or ramped direct-current amperometry can, however, provide current-voltage (I/V) data that may be used to measure the voltage dependency of the membrane conductance. We show that potential gradients >∼150 mV induce membrane defects that permit the insertion of pore-forming peptides. Further, we report here the novel (to our knowledge) use of real-time modeling of conventional low-voltage alternating-current impedance spectroscopy to identify whether the conduction arising from the insertion of a polypeptide is uniform or heterogeneous on scales of nanometers to micrometers across the membrane. The utility of this tBLM architecture and these techniques is demonstrated by characterizing the resulting conduction properties of the antimicrobial peptide PGLa.

Fluorinated amino acids can have dramatic effects on protein stability and protein–protein interactions due to the unique stereoelectronic properties of fluorine. Previous approaches to assessing their properties have mainly focused on helical systems, even though fluoro-amino acids are known to exhibit lower intrinsic helix propensities than their hydrocarbon analogues. Fluorination of specific β-sheet positions within globular proteins has been shown to have a stabilizing effect, suggesting that fluorinated amino acids may generally be well suitable for modulating non-helical structures. Still, fluorinated amino acids have rarely been studied in amyloid forming peptides, which take on a characteristically high cross-β-sheet content. Here, we examine the substitution of natural amino acids within an amyloid forming model peptide by amino acids that contain different stoichiometries of fluorine in their side chains. This approach enables a systematic evaluation of the impact of fluorine on amyloid formation. We have investigated the impact of size, hydrophobicity and secondary structure propensities of the fluorinated amino acids on the amyloid formation process. The structure of the model peptide is based on an engineered coiled coil folding motif that was designed to provide an α-helical starting structure that can fold into β-sheet rich amyloids under controlled conditions. Substitution with fluorinated amino acids was accomplished for two neighboring valine residues that play a key role in the structural transition. The resulting peptides show an unexpected folding behavior as a consequence of the interplay of stereoelectronic effects, helix propensity, hydrophobicity and position of the particular substitution within the amyloid forming system.

Trans -4,5-Difluoromethano-proline was incorporated into the cyclic antimicrobial peptide gramicidin S in place of a native proline residue. Introduction of this intrinsically unstable amino acid into the polypeptide backbone was achieved using a dipeptide strategy. The stable dipeptide building block with the N-acylated 4,5-difluoromethano-proline fragment was obtained by direct difluorocyclopropanation of an unsaturated precursor. The influence of the unnatural amino acid on the conformation and function of gramicidin S was evaluated using circular dichroism and biological assays. The application of trans -4,5-difluoromethano-proline as a new label for solid state 19 F NMR structure analysis of membrane-active peptides was tested on gramicidin S and compared with previous labeling schemes.

We propose a concept for the folding and self- assembly of the pore-forming TatA complex from the Twin-arginine translocase and of other mem- brane proteins based on electrostatic ''charge zippers.'' Each subunit of TatA consists of a trans- membrane segment, an amphiphilic helix (APH), and a C-terminal densely charged region (DCR). The sequence of charges in the DCR is complemen- tary to the charge pattern on the APH, suggesting that the protein can be ''zipped up'' by a ladder of seven salt bridges. The length of the resulting hairpin matches the lipid bilayer thickness, hence a trans- membrane pore could self-assemble via intra- and intermolecular salt bridges. The steric feasibility was rationalized by molecular dynamics simulations, and experimental evidence was obtained by moni- toring the monomer-oligomer equilibrium of specific charge mutants. Similar ''charge zippers'' are pro- posed for other membrane-associated proteins, e.g., the biofilm-inducing peptide TisB, the human antimicrobial peptide dermcidin, and the pestiviral ERNS protein.

Tat signal peptides provide the key signature for proteins that get exported by the bacterial twin arginine translocase. We have characterized the structure of the PhoD signal peptide from Bacillus subtilis in suitable membrane-mimicking environments. High-resolution 13C/15N NMR analysis in detergent micelles revealed a helical stretch in the signal peptide between positions 5 and 15, in good agreement with secondary structure prediction and circular dichroism results. This helix was found to be aligned parallel to the membrane surface according to oriented circular dichroism experiments carried out with planar lipid bilayers. The N-terminal α-helix exhibits a pronounced amphiphilic character, in contrast to the general view in the literature. So far, signal sequences had been supposed to consist of a positively charged N-terminal domain, followed by an α-helical hydrophobic segment, plus a C-terminal domain carrying the peptidase cleavage site. Based on our new structural insights, we propose a model for the folding and membrane interactions of the Tat signal sequence from PhoD.

A new approach for distance measurements in biological solids employing 2 H{ 19 F} rotational echo double resonance was developed and validated on 2 H, 19 F-D-alanine and an imidazopyridine based inhibitor of the gastric H þ /K þ -ATPase. The 2 H– 19 F double resonance experiments presented here were performed without 1 H decoupling using a double resonance NMR spectrometer. In this way, it was possible to benefit from the relatively longer distance range of fluorine without the need of specialized fluorine equipment. A distance of 2.5 � 0.3 � A was measured in the alanine derivative, indicating a gauche conformation of the two labels. In the case of the imidazopyridine compound a lower distance limit of 5.2 � was determined and is in agreement with an extended conformation of the inhibitor. Several REDOR variants were compared, and their advantages and limitations discussed. Composite fluorine dephasing pulses were found to enhance the frequency bandwidth significantly, and to reduce the dependence of the performance of the experiment on the exact choice of the transmitter frequency. 2003 Elsevier Inc. All rights reserved.

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 β-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.

Many solid-state nuclear magnetic resonance (NMR) approaches for membrane proteins rely on orientation-dependent parameters, from which the alignment of peptide segments in the lipid bilayer can be calculated. Molecules embedded in liquid-crystalline membranes, such as monomeric helices, are highly mobile, leading to partial averaging of the measured NMR parameters. These dynamic effects need to be taken into account to avoid misinterpretation of NMR data. Here, we compare two common NMR approaches: (2)H-NMR quadrupolar waves, and separated local field (15)N-(1)H polarization inversion spin exchange at magic angle (PISEMA) spectra, in order to identify their strengths and drawbacks for correctly determining the orientation and mobility of α-helical transmembrane peptides. We first analyzed the model peptide WLP23 in oriented dimyristoylphosphatidylcholine (DMPC) membranes and then contrasted it with published data on GWALP23 in dilauroylphosphatidylcholine (DLPC). We only obtained consistent tilt angles from the two methods when taking dynamics into account. Interestingly, the two related peptides differ fundamentally in their mobility. Although both helices adopt the same tilt in their respective bilayers (~20°), WLP23 undergoes extensive fluctuations in its azimuthal rotation angle, whereas GWALP23 is much less dynamic. Both alternative NMR methods are suitable for characterizing orientation and dynamics, yet they can be optimally used to address different aspects. PISEMA spectra immediately reveal the presence of large-amplitude rotational fluctuations, which are not directly seen by (2)H-NMR. On the other hand, PISEMA was unable to define the azimuthal rotation angle in the case of the highly dynamic WLP23, though the helix tilt could still be determined, irrespective of any dynamics parameters.

Structural parameters of peptides and proteins in biomembranes can be directly measured by solid state NMR of selectively labeled amino acids. The 19F nucleus is a promising label to overcome the low sensitivity of 2H, 13C or 15N, and to serve as a background-free reporter group in biological compounds. To make the advantages of solid state 19F NMR fully available for structural studies of polypeptides, we have systematically measured the chemical shift anisotropies and relaxation properties of the most relevant aromatic and aliphatic 19F-labeled amino acids. In this first part of two consecutive contributions, six different 19F-substituents on representative aromatic side chains were characterized as polycrystalline powders by static and MAS experiments. The data are also compared with results on the same amino acids incorporated in synthetic peptides. The spectra show a wide variety of lineshapes, from which the principal values of the CSA tensors were extracted. In addition, temperature-dependent T(1) and T(2) relaxation times were determined by 19F NMR in the solid state, and isotropic chemical shifts and scalar couplings were obtained in solution.

NMR distance measurements lie at the heart of structural biology. However, long-range distances could not yet be detected in liquid-crystalline biomembranes, because dipolar couplings are partially averaged by the intrinsic molecular mobility. Using conformationally constrained (19)F-labeled amino acids as reporter groups, we could more than double the accessible interatomic distance range by combining a highly sensitive solid-state multipulse (19)F-NMR scheme with a favorable sample geometry. Two rigid 4F-phenylglycine labels were placed into the helical antimicrobial peptide PGLa embedded in fluid oriented membrane samples. A modified Carr-Purcell-Meiboom-Gill sequence yielded an intramolecular distance of 6.6 Å for the labels spanning one helix turn, and 11.0 Å was obtained when the labels spanned two turns. This approach should now also allow the characterization of conformational changes in membrane-active peptides and of oligomeric assemblies in a biologically relevant lipid environment.

Dipolar couplings contain information on internuclear distances as well as orientational constraints. To characterize the structure of the antimicrobial peptide gramicidin S when bound to model membranes, two rigid 4-CF 3 -phenylglycine labels were attached to the cyclic backbone such that they reflect the behavior of the entire peptide. By solid state 19 F NMR we measured the homonuclear dipolar couplings of the two trifluoromethyl-groups in oriented membrane samples. Using the CPMG experiment, both the strong couplings within each CF 3 -group as well as the weak coupling between the two CF 3 -groups could be detected. An intra -CF 3 -group dipolar coupling of 86 Hz and a weak inter -group coupling of 20 Hz were obtained by lineshape simulation of the complex dipolar spectrum. It is thus possible to explore the large distance range provided by 19 F-labels and to resolve weak dipolar couplings even in the presence of strong intra -CF 3 couplings. We applied this approach to distinguish and assign two epimers of the labeled gramicidin S peptide on the basis of their distinct 19 F dipolar coupling patterns.

Membrane composition is a key factor that regulates the destructive activity of antimicrobial peptides and the non-leaky permeation of cell penetrating peptides in vivo. Hence, the choice of model membrane is a crucial aspect in NMR studies and should reflect the biological situation as closely as possible. Here, we explore the structure and dynamics of the short multifunctional peptide BP100 using a multinuclear solid-state NMR approach. The membrane alignment and mobility of this 11 amino acid peptide was studied in various synthetic lipid bilayers with different net charge, fluidity, and thickness, as well as in native biomembranes harvested from prokaryotic and eukaryotic cells. (19)F-NMR provided the high sensitivity and lack of natural abundance background that are necessary to observe a labelled peptide even in protoplast membranes from Micrococcus luteus and in erythrocyte ghosts. Six selectively (19)F-labeled BP100 analogues gave remarkably similar spectra in all of the macroscopically oriented membrane systems, which were studied under quasi-native conditions of ambient temperature and full hydration. This similarity suggests that BP100 has the same surface-bound helical structure and high mobility in the different biomembranes and model membranes alike, independent of charge, thickness or cholesterol content of the system. (31)P-NMR spectra of the phospholipid components did not indicate any bilayer perturbation, so the formation of toroidal wormholes or micellarization can be excluded as a mechanism of its antimicrobial or cell penetrating action. However, (2)H-NMR analysis of the acyl chain order parameter profiles showed that BP100 leads to considerable membrane thinning and thereby local destabilization.

The homonuclear dipolar coupling between the three equivalent 19 F-spins of a trifluoromethyl group, rotating about its threefold symmetry axis, was studied by multipulse solid-state NMR. A modified CPMG sequence was used first to resolve the dipolar splitting of a powder sample, and then to follow its orientation-dependence in uniaxially aligned samples. Our aim is to employ the CF 3 -group as a highly sensitive reporter to describe the mobility and spacial alignment of 19 F-labeled molecules in biomembranes. As an example, the fluorinated anti-inflammatory drug, flufenamic acid, was embedded as a guest compound in lipid bilayers. Undistorted 19 F dipolar spectra of its CF 3 -group were obtained without 1 H-decoupling, revealing a sharp triplet lineshape. When an oriented membrane sample was tilted in the magnetic field, the change in dipolar splittings confirmed that the guest molecule is motionally averaged about the membrane normal, as expected. A different behavior of flufenamic acid, however, was observed under conditions of low bilayer hydration. From this set of orientation-dependent lineshapes we conclude that the axis of motional averaging becomes aligned perpendicular to the sample normal. It thus appears that flufenamic acid induces a hexagonal phase in the membrane at low hydration. Finally, the dipolar 19 F NMR experiments were extended to frozen samples, where no molecular diffusion occurs besides the fast rotation about the CF 3 -axis. Also under these conditions, the CPMG experiment with composite pulses could successfully resolve the dipolar coupling between the three 19 F-nuclei.

Established solid state 31P-NMR and novel 19F-NMR experiments are used in a complementary approach to describe the behaviour of a fluorinated drug, flufenamic acid (FFA), in phospholipid model membranes. The non-steroidal anti-inflammatory agent FFA was dissolved at 5% (w/w) in dimyristoylphosphatidylcholine (DMPC), and the system was investigated at low hydration (3 H2O per lipid) where morphological transitions of the lipid are strongly affected by additives. It is demonstrated that FFA induces a fluid HII phase in DMPC at ambient temperatures, i.e. much below its regular chain-melting transition which occurs around 50°C at low hydration. The guest molecules are preferentially accommodated in the hexagonal phase of the lipid, which coexists with the usual crystalline state of pure DMPC. The peculiar transition sequence LC→HII→Lα with increasing temperature is explained by a re-distribution of FFA in the lipid matrix and a concomitant phase separation under conditions of limiting hydration. Small-angle X-ray diffraction and freeze–fracture electron microscopy are used to confirm the existence of the hexagonal and bilayer phases, and to determine their respective dimensions. When the drug FFA dissolves in the bilayer, its structural effect on the surrounding lipid molecules may be related to its pharmacological activity in membranes. For example, FFA is known to modulate ion channel function, and it has been suggested that it inhibits phospholipase activity by accelerating the transbilayer flip-flop of lipids.

Local macromolecular structure can be determined by solid-state NMR measurements of weak dipolar couplings between selectively labeled groups. The nonperturbing use of 2 H, 13 C, or 15 N in biological systems, however, faces drawbacks in terms of a low sensitivity and a comparatively short distance range relative to 1 H. To extend these limitations, we illustrate the use of 19 F as an alternative NMR probe. The Carr–Purcell–Meiboom–Gill (CPMG) multipulse sequence was adapted here to measure homonuclear dipolar couplings between two fluorine labels in static samples at 470 MHz. Two lipids (4,4-DMPC-F 2 , and a difluorinated sterol), which are arranged in liquid crystalline bilayers, serve as models to assess the scope of the technique. In these 19 F-background-free biological samples, weak couplings down to 100 Hz could be resolved directly from the splitting of the pure dipolar powder lineshape, and 1 H-decoupling was not required. Order parameters were determined for the anisotropic motion of the lipids, consistent with their expected behavior in the membrane. Besides measuring the distance-dependent term of the dipolar coupling in powder samples, we have also used oriented membranes to extract additional angular information from the dipolar anisotropy. The strategy presented here thus has the potential to obtain not only the internuclear distance between two labels, but also their angular orientation in the sample, provided the molecules are aligned as a membrane or a fiber.

The alignment of the antimicrobial peptide PGLa in a lipid bilayer was characterized by solid state 19F NMR on selectively CF3-labeled peptides in oriented samples. Previous studies in liquid crystalline model membranes had shown that the amphiphilic α-helix of PGLa adopts a surface-aligned S-state or a tilted T-state, depending on the peptide/lipid ratio. Only in the presence of the synergistic partner peptide magainin 2 had PGLa been found insert into the bilayer in a transmembrane I-state orientation. Here, we have characterized the peptide alignment but as a function of temperature and lipid phase state. At temperatures below the lipid chain melting transition, PGLa is now seen to be able to insert on its own, with its helical axis nearly parallel to the bilayer normal (tilt angle of ∼170°), forming the I-state. Above the lipid phase transition, PGLa is aligned in the known T-state (tilt angle of ∼130°), but it is found flip into the S-state at more elevated temperatures (tilt angle of ∼96°). This way...

Many vital cellular processes, such as protein translocation, proton transport or molecular recognition, are mediated by self assembling membrane proteins. We have investigated the Twin-arginine translocase (TatA) complex, which forms transient pores through which proteins are translocated through the membrane. We postulated that complex formation is electrostatically driven by formation of salt bridges between amphiphilic transmembrane segments of the individual monomers and developed a structure-based model for this process[1].We studied the formation of oligomers of different sizes by structure-based[2] MD simulations in combination with NMR constraints and a hydrophobic-slab implicit membrane model. Starting from isolated monomers, distributed far apart from each other, we observed the formation of stable TatA oligomers on the basis of the postulated interactions. The dimensions of the resulting TatA complex agreed well with experimental electron microscopy measurements[3] and the postulated interactions were confirmed by subsequent mutation studies.[1] T. Walther, C. Gottselig, S. Grage, M. Wolf, A. Vargiu, Marco J. Klein, S. Vollmer, S. Prock, M. Hartmann, S. Afonin, E. Stockwald, H. Heinzmann, W Wenzel, P. Ruggerone, A. Ulrich. Folding and self-assembly of the TatA translocation pore based on a novel charge zipper mechanism, Cell (accepted)[2] Whitford, P. C. et al. An all-atom structure-based potential for proteins: Bridging minimal models with all-atom empirical forcefields. Proteins 75, 430-441 (2009).[3] Gohlke, U. et al. The TatA component of the twin-arginine protein transport system forms channel complexes of variable diameter. Proc. Natl. Acad. Sci. U.S.A. 102, 10482-10486 (2005).

We describe new techniques to study the insertion of pore forming antimicrobial peptides (AMPs) into tethered bilayer lipid membranes (tBLMs). A consequence of tethering a membrane to a gold surface is that electrical contact to the PBS bathing solution is intrinsically capacitive, preventing the direct application of a steady-state voltage across the bilayer. However, by using pulsed waveforms, defined potentials may be expressed across the membrane for tens to hundreds of milliseconds, and the resulting I-V plots provide valuable data about AMP insertion rates and voltage dependence (Fig 1).Using this technique in the presence of PGLa, we demonstrate how AMP insertion into zwitterionic and negatively charged lipid membranes can be rapidly measured and compared. To better understand the voltage dependence of AMP insertion into tBLMs, ramped potentials can also be applied which can determine the potential thresholds of peptide insertion and pore formation.Advantages of using tBLMs:• tBLMs are more robust and longer lasting than black lipid membranes, or micropipette patches;• Easier, quicker sample preparation than conventional voltage clamp experiments;• Physiologically relevant AMP concentrations can be used (cf. NMR).View Large Image | View Hi-Res Image | Download PowerPoint Slide

In biological membranes the alignment of embedded proteins provides crucial structural information. The transmembrane (TM) parts have well-defined secondary structures, in most cases α-helices and their orientation is given by a tilt angle and an azimuthal rotation angle around the main axis. The tilt angle is readily visualized and has been found to be functionally relevant. However, there exist no general concepts on the corresponding azimuthal rotation. Here, we show that TM helices prefer discrete rotation angles. They arise from a combination of intrinsic properties of the helix geometry plus the influence of the position and type of flanking residues at both ends of the hydrophobic core. The helical geometry gives rise to canonical azimuthal angles for which the side chains of residues from the two ends of the TM helix tend to have maximum or minimum immersion within the membrane. This affects the preferential position of residues that fall near hydrophobic/polar interfaces of the membrane, depending on their hydrophobicity and capacity to form specific anchoring interactions. On this basis, we can explain the orientation and dynamics of TM helices and make accurate predictions, which correspond well to the experimental values of several model peptides (including dimers), and TM segments of polytopic membrane proteins.