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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.

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.

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.

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.

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.

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.

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.

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.

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.

Mechanosensitive channels allow bacteria to respond to osmotic stress by opening a nanometer-sized pore in the cellular membrane. Although the underlying mechanism has been thoroughly studied on the basis of individual channels, the behavior of channel ensembles has yet to be elucidated. This work reveals that mechanosensitive channels of large conductance (MscL) exhibit a tendency to spatially cluster, and demonstrates the functional relevance of clustering. We evaluated the spatial distribution of channels in a lipid bilayer using patch-clamp electrophysiology, fluorescence and atomic force microscopy, and neutron scattering and reflection techniques, coupled with mathematical modeling of the mechanics of a membrane crowded with proteins. The results indicate that MscL forms clusters under a wide range of conditions. MscL is closely packed within each cluster but is still active and mechanosensitive. However, the channel activity is modulated by the presence of neighboring proteins, indicating membrane-mediated protein-protein interactions. Collectively, these results suggest that MscL self-assembly into channel clusters plays an osmoregulatory functional role in the membrane.

Many membrane-active antimicrobial peptides are cationic and fold into amphiphilic structures upon binding to the lipid bilayer of the bacterial envelope, which thereupon gets permeabilized. Peptides with an alpha-helical conformation have been thoroughly studied by solid state NMR and other biophysical techniques. A concerted re-alignment of several monomers is supposed to lead to the formation of a transient pore, i.e. a local oligomeric assembly with anionic lipids that is called toroidal wormhole. Yet, several designer-made “helical” peptides have been found to undergo a rapid membrane-induced concentration-dependent aggregation as beta-strands. Given that such H-bonded aggregate is thermodynamically more stable, the question arises whether membrane perturbation involves only the “classical” helical structures under kinetic control, or whether oligomeric beta-sheets may also contribute, as proposed e.g. for the cytotoxic Alzheimer's peptide. Even more intriguing is the same question when referring to designated beta-stranded peptides, such as the (KIGAKI)3 system designed by Blazyk et al. as a complement to the helical (KIAGKIA)3, both of which have comparable antimicrobial activity. We have studied these and other representative antimicrobial peptides with alpha-helical and beta-stranded character using solid state 19F-NMR and circular dichroism. In macroscopically oriented samples, a combination of these two methods can reveal not only the conformation and alignment of a peptide in the lipid bilayer, but also its local dynamic behavior and global aggregation kinetics. Structural results and their correlation with antimicrobial activity will be presented, in an attempt to address the mechanistic questions raised above.

Membrane-active peptides or protein segments play an important role in many biological processes at the cellular interface to the environment. They are involved, e.g., in cellular fusion or host defense, where they can cause not only merging but also the destabilization of cell membranes. Many factors determine how these typically amphipathic peptides interact with the lipid bilayer. For example, the peptide orientation in the membrane determines which parts of the peptide are exposed to the hydrophobic bilayer interior or to the polar lipid/water interface. As another example, oligomerization is required for many activities such as pore formation. Peptides have been often classified according to a single characteristic mode of interaction with the bilayer, but over the years a more versatile picture has emerged. It appears that any single peptide can adopt several different alignments and/or oligomeric states in response to changes in the environment. For instance, many antimicrobial peptides adopt a surface-parallel alignment at low concentration, but they tilt obliquely into or even fully insert transmembrane into the bilayer above a critical peptide-to-lipid ratio, often in the form of oligomeric pores. Similar changes in peptide orientation or oligomeric state have been observed as a function of, e.g., temperature, lipid composition, pH, or induced by a synergistic partner peptide. Such transitions between peptide states can be regarded as the result of a re-adjustment in the balance between peptide-peptide and peptide-lipid interactions, as the environment conditions are changed. Though often studied in model membrane systems, such rich variety of peptide states is even more likely to occur in native biomembranes with their diverse compositions and physicochemical properties. The ability to undergo transitions between different states thus plays a fundamental role for the biological activities of membrane-active peptides.

We have observed a hydrocarbon chain length dependent perturbation of saturated acyl chain phosphatidylglycerol bilayers by the antimicrobial peptide peptidyl-glycylleucine-carboxyamide (PGLa) using X-ray diffraction, solid-state 2H-NMR, differential scanning calorimetry and dilatometry. In the gel phase, PGLa assumes a surface alignment and induces a quasi-interdigitated phase, previously reported also for other peptides. This effect is most pronounced for C18 phosphatidylglycerol. Above the lipid chain melting transition, in the fluid phase, the PGLa helix inserts into the membrane above a certain threshold concentration. In this case we found an increase of the membrane thickness and NMR order parameter for C14 and C16 phosphatidylglycerol bilayers, though not for C18. The data is best understood in terms of a close hydrophobic match between the C18 bilayer core and the peptide length when PGLa is inserted with its helical axis normal to the bilayer surface. The C16 acyl chains appear to stretch in order to accommodate PGLa, whereas tilting within the bilayer seems to be energetically favorable for the peptide when inserted into bilayers of C14 phosphatidylglycerol. In contrast to the commonly accepted membrane thinning effect of antimicrobial peptides, the data demonstrate that pore formation does not necessarily relate to changes in the overall bilayer structure.

Cells have developed sophisticated transport machineries to allow proteins to cross membrane barriers. In bacteria, the twin arginine translocase (Tat) can translocate proteins even in the folded state. In B. subtilis, a membrane protein complex consisting of two subunits, TatA and TatC, is responsible for the Tat translocation process. TatA is believed to form a nanometer size pore trough which the protein is transported, whereas TatC is involved in recognition of the target protein signal peptide.View Large Image | View Hi-Res Image | Download PowerPoint SlideTo get insight into the mechanism of the Tat translocation in B. subtilis, we studied the structure of the pore-forming subunit TatA and the pore assembly pursuing two complementary experimental approaches. The structure of individual TatA monomers in membranes or membrane-mimetic environments was characterized using solid state and solution NMR. The formation of oligomeric assemblies of TatA in the membrane, on the other hand, was investigated using in-plane neutron scattering of TatA reconstituted in aligned membranes. This way, we were able to both derive a detailed structural model of TatA, and to characterize pores formed by TatA.

Several different membrane-active peptides were labeled with a variety of fluorinated amino acids for structure analysis by solid state 19 F NMR. Namely, 4-F-Phg/4-CF 3 -Phg, 3,3,3-F 3 -Ala/3-F-Ala, and 2-CF 3 -Ala were used to replace a single amino acid such as Ile/Leu, Ala, and Aib, respectively, without significantly perturbing the peptide conformation or function. These NMR reporter groups can be analyzed to calculate the structure and mobility of the peptide in the lipid bilayer. This review focuses on synthetic challenges with 19 F-labeled amino acids, such as racemization and fluorine elimination, and recent results on various antimicrobial and fusogenic peptides in model membranes will be summarized.

Knowledge of structural details at an atomic level is important in understanding the function and molecular properties of biomolecules. In situations where crystallography or solution state NMR methods are not applicable, solid state NMR can provide valuable insight without the limitations imposed by the availability of crystals, solubility or molecular weight. In particular dedicated solid state NMR methods enable the determination of selective internuclear distances at high accuracy. A popular and robust technique is rotational echo double resonance (REDOR), which allows the measurement of distances of heteronuclear spin pairs. This magic angle spinning method uses radiofrequency pulses to prevent the dipole-dipole interaction from being averaged by the sample spinning, leading to a reduction of the NMR signal which depends on the internuclear distance. Biological systems constitute a major area of application of the REDOR experiment, and are the focus of this review. First, the theoretical background, developments and experimental considerations of REDOR are discussed, regarding in particular applications on biological samples. Then, an overview of the use of REDOR in a wide range of biological applications, such as ligand binding sites of enzymes, fibrilar proteins, membrane active peptides, transmembrane helices, biomaterials like the peptidoglycan of bacterial cell walls or nucleic acids, is presented. © 2007 Elsevier Ltd. All rights reserved.

The interaction of the fusogenic polypeptide segment "B18" from the fertilization protein binding with lipid membranes was investigated by solid state 2H and 31P NMR, and by differential scanning calorimetry. B18 is known to adopt different conformations depending on peptide concentration, ionic conditions, pH and lipid environment. Here, the peptide was studied in its beta-stranded amyloid conformation. According to 31P NMR, the lamellar morphology of the DMPC bilayer remains intact in the presence of B18. In going from low (1:90) to high (1:10) peptide/lipid ratios, an increasing effect on several different 2H-labeled lipid segments was observed, reflecting changes in phase behavior and local dynamics. The strongest influence of B18 was detected at the acyl-chains, while no significant effect on the lipid headgroup conformation was observed. This suggests an insertion of B18 in its fibrillar state into the membrane driven by hydrophobic interactions, rather than a peripheral binding mediated by electrostatics.

Solid-state NMR is emerging as a method for resolving structural information for large biomolecular complexes, such as membrane-embedded proteins. In principle, there is no molecular weight limit to the use of the approach, although the complexity and volume of data is still outside complete assignment and structural determinations for any large (Mr > approx 30,000) complex unless specific methods to reduce the information content to a manageable amount are employed. Such methods include specific residue-type labeling, labeling of putative segments of a protein, or examination of complexes made up of smaller, manageable units, such as oligomeric ion channels. Labeling possibilities are usually limited to recombinant or synthesized proteins, and labeling strategies often follow models from a bioinformatics approach. In all cases, and in common with most membrane studies, sample preparation is vital, and this activity alone can take considerable effort before NMR can be applied--peptide or protein production (synthesis or expression) followed by reconstitution into bilayers and resolution of suitable sample geometry is still technically challenging. As experience is gained in the field, this development time should decrease. Here, the practical aspects of the use of solid-state NMR for membrane protein structural determinations are presented, as well as how the methodology can be applied. Some successes to date are discussed, with an indication of how the area might develop.

Structure determination of membrane-associated polypeptides presents one of the major challenges to solid state NMR spectroscopy. Many studies have been carried out so far using selective isotope labels, such as 2H, 13C, or 15N[1]. These NMR-reporters can be incorporated into the protein backbone or side chains, to reveal local structural parameters and to describe the dynamic properties of the membrane-embedded molecule. For example, an distance r can be measured between a pair of labels by means of dipolar recoupling MAS techniques such as rotational resonance or REDOR[2]. Alternatively, uniaxially oriented samples are used to determine the angle A of a labelled molecular segment with respect to the membrane normal N[3,4,5]. The latter approach relies on the orientation-dependent resonance frequency, which carries information about the anisotropie chemical shift tensor (13C, 15N), the dipolar coupling (1H-15N), or the quadrupolar interaction (2H)[6,7].

The bacterial Twin-arginine translocation pathway is able to transport fully folded proteins across membranes. In B. subtilis it consists only of two components: TatCd, which serves as a receptor for the signal peptide, and the pore forming unit TatAd, which occurs in high stoichiometric excess. According to circular dichroism TatA contains a transmembrane segment, an amphiphilic helix, and an unstructured C-terminus [1]. Its detailed moelcular structure was resolved by solid-state NMR spectroscopy in oriented bilayers [2]. A striking pattern on the monomeric protein surface allowed us to assemble several units into protomers and into an open oligomeric pore. The stability of these complexes was supported by all-atom MD simulations and using structure-based modeling [3]. The observed interactions suggest that a novel motif for folding and self-assembly motif is present in this membrane-bound transport system, which allows reversible pore formation. Our comprehensive three-dimensional model thus reconciles for the first time TatA transport with a pore size of variable diameter, which can open and close by an energetically feasible mechanism.[1] Muller, S.D., A.A. De Angelis, T.H. Walther, S.L. Grage, C. Lange, S.J. Opella & A.S. Ulrich (2007) Biochim. Biophys. Acta 1768: 3071-3079[2] Walther, T.H., S.L. Grage, N. Roth & A.S. Ulrich (2010) J. Am. Chem. Soc., in press[3] Grage, S.L., T.H. Walther, M. Wolf, A. Vargiu, M.J. Klein, P. Ruggerone, W. Wenzel, A.S. Ulrich (2010) submitted∼

The response of membrane-associated peptides toward the lipid environment or other binding partners can be monitored by solid-state NMR of suitably labeled side chains. Tryptophan is a prominent amino acid in transmembrane helices, and its (19)F-labeled analogues are generally biocompatible and cause little structural perturbation. Hence, we use 5F-Trp as a highly sensitive NMR probe to monitor the conformation and dynamics of the indole ring. To establish this (19)F-NMR strategy, gramicidin A was labeled with 5F-Trp in position 13 or 15, whose chi(1)/chi(2) torsion angles are known from previous (2)H-NMR studies. First, the alignment of the (19)F chemical shift anisotropy tensor within the membrane was deduced by lineshape analysis of oriented samples. Next, the three principal axes of the (19)F chemical shift anisotropy tensor were assigned within the molecular frame of the indole ring. Finally, determination of chi(1)/chi(2) for 5F-Trp in the lipid gel phase showed that the side chain alignment differs by up to 20 degrees from its known conformation in the liquid crystalline state. The sensitivity gain of (19)F-NMR and the reduction in the amount of material was at least 10-fold compared with previous (2)H-NMR studies on the same system and 100-fold compared with (15)N-NMR.

Using x-ray diffraction, solid-state 2H-NMR, differential scanning calorimetry, and dilatometry, we have observed a perturbation of saturated acyl chain phosphatidylglycerol bilayers by the antimicrobial peptide peptidyl-glycylleucine-carboxyamide (PGLa) that is dependent on the length of the hydrocarbon chain. In the gel phase, PGLa induces a quasi-interdigitated phase, previously reported also for other peptides, which is most pronounced for C18 phosphatidylglycerol. In the fluid phase, we found an increase of the membrane thickness and NMR order parameter for C14 and C16 phosphatidylglycerol bilayers, though not for C18. The data is best understood in terms of a close hydrophobic match between the C18 bilayer core and the peptide length when PGLa is inserted with its helical axis normal to the bilayer surface. The C16 acyl chains appear to stretch to accommodate PGLa, whereas tilting within the bilayer seems to be energetically favorable for the peptide when inserted into bilayers of C14 phosphatidylglycerol. In contrast to the commonly accepted membrane thinning effect of antimicrobial peptides, the data demonstrate that pore formation does not necessarily relate to changes in the overall bilayer structure.

The transmembrane protein TatA is the pore forming unit of the twin-arginine translocase (Tat), which has the unique ability of transporting folded proteins across the cell membrane. This ATP-independent protein export pathway is a recently discovered alternative to the general secretory (Sec) system of bacteria. To obtain insight in the translocation mechanism, the structure and alignment in the membrane of the well-folded segments 2–45 of TatAd from Bacillus subtilis was studied here. Using solid-state NMR in bicelles containing anionic lipids, the topology and orientation of TatAd was determined in an environment mimicking the bacterial membrane. A wheel-like pattern, characteristic for a tilted transmembrane helix, was observed in 15N chemical shift /15N–1H dipolar coupling correlation NMR spectra. Analysis of this PISA wheel revealed a 14–16 residue long N-terminal membrane-spanning helix which is tilted by 17° with respect to the membrane normal. In addition, comparison of uniformly and selectively 15N-labeled TatA2–45 samples allowed determination of the helix polarity angle.