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Antimicrobial peptides are an important class of membrane-active peptides that can provide alternatives or complements to classic antibiotics. Among the many classes of AMPs, the histidine-rich family is of particular interest since they may induce pH-sensitive interactions with cell membranes. The AMP caerin 1.1 (Cae-1), from Australian tree frogs, has three histidine residues, and thus we studied the pH dependence of its interactions with model cell membranes. Using NMR spectroscopy and molecular dynamics simulations, we showed that Cae-1 induced greater perturbation of the lipid dynamics and water penetrations within the membrane interior in an acidic environment compared with physiological conditions. Using 31 P solid-state NMR, the packing, chemical environment, and dynamics of the lipid headgroup were monitored. 2 H solid-state NMR showed that Cae-1 ordered the acyl chains of the hydrophobic core of the bilayer. These results supported the molecular dynamics data, which showed that Cae-1 was mainly inserted within the lipid bilayer for both neutral and negatively charged membranes, with the charged residues pulling the water and phosphate groups inward. This could be an early step in the mechanism of membrane disruption by histidine-rich antimicrobial peptides and indicated that Cae-1 acts via a transmembrane mechanism in bilayers of neutral and anionic phospholipid membranes, especially in acidic conditions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The benefit of combining in-cell solid-state dynamic nuclear polarization (DNP) NMR and cryogenic temperatures is providing sufficient signal/noise and preservation of bacterial integrity via cryoprotection to enable in situ biophysical studies of antimicrobial peptides. The radical source required for DNP was delivered into cells by adding a nitroxide-tagged peptide based on the antimicrobial peptide maculatin 1.1 (Mac1). In this study, the structure, localization, and signal enhancement properties of a single (T-MacW) and double (T-T-MacW) TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) spin-labeled Mac1 analogs were determined within micelles or lipid vesicles. The solution NMR and circular dichroism results showed that the spin-labeled peptides adopted helical structures in contact with micelles. The peptides behaved as an isolated radical source in the presence of multilamellar vesicles, and the electron paramagnetic resonance (EPR) electron-electron distance for the doubly spin-labeled peptide was ∼1 nm. The strongest paramagnetic relaxation enhancement (PRE) was observed for the lipid NMR signals near the glycerol-carbonyl backbone and was stronger for the doubly spin-labeled peptide. Molecular dynamics simulation of the T-T-MacW radical source in phospholipid bilayers supported the EPR and PRE observations while providing further structural insights. Overall, the T-T-MacW peptide achieved better 13 C and 15 N signal NMR enhancements and 1 H spin-lattice T 1 relaxation than T-MacW., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Resistance to the last-resort antibiotic colistin is now widespread and new therapeutics are urgently required. We report the first in toto chemical synthesis and pre-clinical evaluation of octapeptins, a class of lipopeptides structurally related to colistin. The octapeptin biosynthetic cluster consisted of three non-ribosomal peptide synthetases (OctA, OctB, and OctC) that produced an amphiphilic antibiotic, octapeptin C4, which was shown to bind to and depolarize membranes. While active against multi-drug resistant (MDR) strains in vitro, octapeptin C4 displayed poor in vivo efficacy, most likely due to high plasma protein binding. Nuclear magnetic resonance solution structures, empirical structure-activity and structure-toxicity models were used to design synthetic octapeptins active against MDR and extensively drug-resistant (XDR) bacteria. The scaffold was then subtly altered to reduce plasma protein binding, while maintaining activity against MDR and XDR bacteria. In vivo efficacy was demonstrated in a murine bacteremia model with a colistin-resistant P. aeruginosa clinical isolate., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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 19 F-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 19 F-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 19 F-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 15 N-NMR of uniformly labeled protein, and confirmed using solid-state 31 P- and 2 H-NMR. 31 P-NMR relaxation data indicated a change in lipid headgroup dynamics induced by kalata B1. Changes in the 2 H-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 19 F-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., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Antimicrobial peptides (AMPs) are a class of host-defense molecules that neutralize a broad range of pathogens. Their membrane-permeabilizing behavior has been commonly attributed to the formation of pores; however, with the continuing discovery of AMPs, many are uncharacterized and their exact mechanism remains unknown. Using atomic force microscopy, we previously characterized the disruption of model membranes by protegrin-1 (PG-1), a cationic AMP from pig leukocytes. When incubated with zwitterionic membranes of dimyristoylphosphocholine, PG-1 first induced edge instability at low concentrations, then porous defects at intermediate concentrations, and finally worm-like micelle structures at high concentrations. These rich structural changes suggested that pore formation constitutes only an intermediate state along the route of PG-1's membrane disruption process. The formation of these structures could be best understood by using a mesophase framework of a binary mixture of lipids and peptides, where PG-1 acts as a line-active agent in lowering interfacial bilayer tensions. We have proposed that rather than being static pore formers, AMPs share a common ability to lower interfacial tensions that promote membrane transformations. In a study of 13 different AMPs, we found that peptide line-active behavior was not driven by the overall charge, and instead was correlated with their adoption of imperfect secondary structures. These peptide structures commonly positioned charged residues near the membrane interface to promote deformation favorable for their incorporation into the membrane. Uniquely, the data showed that barrel-stave-forming peptides such as alamethicin are not line-active, and that the seemingly disparate models of toroidal pores and carpet activity are actually related. We speculate that this interplay between peptide structure and the distribution of polar residues in relation to the membrane governs AMP line activity in general and represents a novel, to our knowledge, avenue for the rational design of new drugs., (Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Equinatoxin II (EqtII) is a soluble, 20 kDa pore-forming protein toxin isolated from the sea anemone Actinia equina. Although pore formation has long been known to occur in distinct stages, including monomeric attachment to phospholipid membranes followed by detachment of the N-terminal helical domain and oligomerization into the final pore assembly, atomistic-level detail of the protein-lipid interactions underlying these events remains elusive. Using high-resolution solution state NMR of uniformly-(15)N-labeled EqtII at the critical micelle concentration of dodecylphosphocholine, we have mapped the lipid-binding site through chemical shift perturbations. Subsequent docking of an EqtII monomer onto a dodecylphosphocholine micelle, followed by 400 ns of all-atom molecular dynamics simulation, saw several high-occupancy lipid-binding pockets stabilized by cation-π, hydrogen bonding, and hydrophobic interactions; and stabilization of the loop housing the conserved arginine-glycine-aspartate motif. Additional simulation of EqtII with an N-acetyl sphingomyelin micelle, for which high-resolution NMR data cannot be obtained due to aggregate formation, revealed that sphingomyelin specificity might occur via hydrogen bonding to the 3-OH and 2-NH groups unique to the ceramide backbone by side chains of D109 and Y113; and main chains of P81 and W112. Furthermore, a binding pocket formed by K30, K77, and P81, proximate to the hinge region of the N-terminal helix, was identified and may be implicated in triggering pore formation., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The role of proline in the disruption of membrane bilayer structure upon antimicrobial peptide (AMP) binding was studied. Specifically, (31)P and (2)H solid-state NMR and dual polarization interferometry (DPI) were used to analyze the membrane interactions of three AMPs: maculatin 1.1 and two analogs in which Pro-15 is replaced by Gly and Ala. For NMR, deuterated dimyristoylphosphatidylcholine (d54-DMPC) and d54-DMPC/dimyristoylphosphatidylglycerol (DMPG) were used to mimic eukaryotic and prokaryotic membranes, respectively. In fluid-phase DMPC bilayer systems, the peptides interacted primarily with the bilayer surface, with the native peptide having the strongest interaction. In the mixed DMPC/DMPG bilayers, maculatin 1.1 induced DMPG phase separation, whereas the analogs promoted the formation of isotropic and lipid-enriched phases with an enhanced effect relative to the neutral DMPC bilayers. In gel-phase DMPC vesicles, the native peptide disrupted the bilayer via a surface mechanism, and the effect of the analogs was similar to that observed in the fluid phase. Real-time changes in bilayer order were examined via DPI, with changes in bilayer birefringence analyzed as a function of the peptide mass bound to the bilayer. Although all three peptides decreased the bilayer order as a function of bound concentration, maculatin 1.1 caused the largest change in bilayer structure. The NMR data indicate that maculatin 1.1 binds predominantly at the surface regions of the bilayer, and both NMR and DPI results indicate that this binding leads to a drop in bilayer order. Overall, the results demonstrate that the proline at residue 15 plays a central role in the membrane interaction of maculatin 1.1 by inducing a significant change in membrane order and affecting the ability of the bilayer to recover from structural changes induced by the binding and insertion of the peptide., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Static phosphorous NMR has been a powerful technique for the study of model supramolecular phospholipid structures. Application to natural lipid bilayers with complex compositions, however, has been severely limited by the difficulty in deconvoluting overlapping broad lineshapes. We demonstrate a solution to this problem, using a global fit to a few slow magic-angle spinning spectra, in combination with an adaptation of Boltzmann statistics maximum entropy. The method provides a model-free means to characterize a heterogeneous mix of lipid dynamics via a distribution of (31)P chemical shift anisotropies. It is used here to identify clear changes in membrane dynamics of a phosphatidylethanolamine and phosphatidylglycerol mixture, mimicking an Escherichia coli membrane upon addition of just 2% of the antimicrobial peptide maculatin 1.1. This illustration opens the prospect for investigation of arbitrarily complex natural lipid systems, important in many areas of biophysical chemistry and biomedicine., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The skin secretions of Australian tree frogs are rich in peptides with potential antimicrobial activity. They interrupt bacterial cell membranes, although precisely how and whether all peptides have the same mechanism is not known. The interactions of three of these peptides-aurein 1.2, maculatin 1.1, and caerin 1.1 with supported phospholipid bilayers-are examined here using quartz crystal microbalance and atomic force microscopy. These approaches enabled us to reveal variations in material structure and density as a function of distance from the sensor surface when comparing mass sensorgrams over a range of harmonics of the natural resonance of the sensor crystal and hence obtain for the first time to our knowledge a mechanistic assessment of membrane disruption. We found that caerin inserted into the bilayer in a transmembrane manner, regardless of concentration and phospholipid composition consistent with a pore-forming mechanism. In contrast, maculatin and aurein interacted with membranes in a concentration-dependent manner. At low concentrations (<5 microM), maculatin exhibited transmembrane incorporation whereas aurein was limited to surface association. Upon reaching a threshold value of concentration, both peptides lysed the membrane. In the case of maculatin, the lysis progressed in a slow, concentration-dependent manner, forming mixed micelles, as shown by atomic force microscopy imaging. Aurein-induced lysis proceeded to a sudden disruption, which is consistent with the "carpet" mechanism. Both maculatin and aurein exhibit specificity toward phospholipids and thus have potential as candidates as antimicrobial drugs.

Membrane lysis caused by antibiotic peptides is often rationalized by means of two different models: the so-called carpet model and the pore-forming model. We report here on the lytic activity of antibiotic peptides from Australian tree frogs, maculatin 1.1, citropin 1.1, and aurein 1.2, on POPC or POPC/POPG model membranes. Leakage experiments using fluorescence spectroscopy indicated that the peptide/lipid mol ratio necessary to induce 50% of probe leakage was smaller for maculatin compared with aurein or citropin, regardless of lipid membrane composition. To gain further insight into the lytic mechanism of these peptides we performed single vesicle experiments using confocal fluorescence microscopy. In these experiments, the time course of leakage for different molecular weight (water soluble) fluorescent markers incorporated inside of single giant unilamellar vesicles is observed after peptide exposure. We conclude that maculatin and its related peptides demonstrate a pore-forming mechanism (differential leakage of small fluorescent probe compared with high molecular weight markers). Conversely, citropin and aurein provoke a total membrane destabilization with vesicle burst without sequential probe leakage, an effect that can be assigned to a carpeting mechanism of lytic action. Additionally, to study the relevance of the proline residue on the membrane-action properties of maculatin, the same experimental approach was used for maculatin-Ala and maculatin-Gly (Pro-15 was replaced by Ala or Gly, respectively). Although a similar peptide/lipid mol ratio was necessary to induce 50% of leakage for POPC membranes, the lytic activity of maculatin-Ala and maculatin-Gly decreased in POPC/POPG (1:1 mol) membranes compared with that observed for the naturally occurring maculatin sequence. As observed for maculatin, the lytic action of Maculatin-Ala and maculatin-Gly is in keeping with the formation of pore-like structures at the membrane independently of lipid composition.

Amyloid aggregates, found in patients that suffer from Alzheimer's disease, are composed of fibril-forming peptides in a beta-sheet conformation. One of the most abundant components in amyloid aggregates is the beta-amyloid peptide 1-42 (Abeta 1-42). Membrane alterations may proceed to cell death by either an oxidative stress mechanism, caused by the peptide and synergized by transition metal ions, or through formation of ion channels by peptide interfacial self-aggregation. Here we demonstrate that Langmuir films of Abeta 1-42, either in pure form or mixed with lipids, develop stable monomolecular arrays with a high surface stability. By using micropipette aspiration technique and confocal microscopy we show that Abeta 1-42 induces a strong membrane destabilization in giant unilamellar vesicles composed of palmitoyloleoyl-phosphatidylcholine, sphingomyelin, and cholesterol, lowering the critical tension of vesicle rupture. Additionally, Abeta 1-42 triggers the induction of a sequential leakage of low- and high-molecular-weight markers trapped inside the giant unilamellar vesicles, but preserving the vesicle shape. Consequently, the Abeta 1-42 sequence confers particular molecular properties to the peptide that, in turn, influence supramolecular properties associated to membranes that may result in toxicity, including: 1), an ability of the peptide to strongly associate with the membrane; 2), a reduction of lateral membrane cohesive forces; and 3), a capacity to break the transbilayer gradient and puncture sealed vesicles.

Enkephalins are pentapeptides found in the central nervous system. It is believed that these neuropeptides interact with the nerve cell membrane to adopt a conformation suitable for their binding to an opiate receptor. In this work, we have determined the three-dimensional structure of methionine-enkephalin (Menk) in fast-tumbling bicelles using multidimensional (1)H NMR. Bicelles were selected as model membranes because both their bilayer organization and composition resemble those of natural biomembranes. The effect of the membrane composition on the peptide conformation was explored using both zwitterionic (PC bicelles) and negatively charged bicelles (Bic/PG). Pulsed field gradient experiments allowed the determination of the proportion of Menk bound to the model membranes. Approximately 60% of the water-soluble enkephalin was found to associate to the bicellar systems. Structure calculations from torsion angle and NOE-based distance constraints suggest the presence of both micro - and delta-selective conformers of Menk in each system and slightly different conformers in PC bicelles and Bic/PG. As opposed to previous studies of enkephalins in membrane mimetic systems, our results show that these opiate peptides could adopt several conformations in a membrane environment, which is consistent with the flexibility and poor selectivity of enkephalins.

Equinatoxin II (EqtII), a protein toxin from the sea anemone Actinia equina, readily creates pores in sphingomyelin-containing lipid membranes. The perturbation by EqtII of model lipid membranes composed of dimyristoylphosphatidycholine and sphingomyelin (10 mol %) was investigated using wideline phosphorus-31 and deuterium NMR. The preferential interaction between EqtII (0.1 and 0.4 mol %) and the individual bilayer lipids was studied by (31)P magic angle spinning NMR, and toxin-induced changes in bilayer morphology were examined by freeze-fracture electron microscopy. Both NMR and EM showed the formation of an additional lipid phase in sphingomyelin-containing mixed lipid multilamellar suspensions with 0.4 mol % EqtII. The new toxin-induced phase consisted of small unilamellar vesicles 20-40 nm in diameter. Deuterium NMR showed that the new lipid phase contains both dimyristoylphosphatidycholine and sphingomyelin. Solid-state (31)P NMR showed an increase in spin-lattice and a decrease in spin-spin relaxation times in mixed-lipid model membranes in the presence of EqtII, consistent with an increase in the intensity of low frequency motions. The (2)H and (31)P spectral intensity distributions confirmed a change in lipid mobility and showed the creation of an isotropic lipid phase, which was identified as the small vesicle structures visible by electron microscopy in the EqtII-lipid suspensions. The toxin appears to enhance slow motions in the membrane lipids and destabilize the membrane. This effect was greatly enhanced in sphingomyelin-containing mixed lipid membranes compared with pure phosphatidylcholine bilayers, suggesting a preferential interaction between the toxin and bilayer sphingomyelin.

Solid-state (13)C NMR spectroscopy was used to investigate the three-dimensional structure of melittin as lyophilized powder and in ditetradecylphosphatidylcholine (DTPC) membranes. The distance between specifically labeled carbons in analogs [1-(13)C]Gly3-[2-(13)C]Ala4, [1-(13)C]Gly3-[2-(13)C]Leu6, [1-(13)C]Leu13-[2-(13)C]Ala15, [2-(13)C]Leu13-[1-(13)C]Ala15, and [1-(13)C]Leu13-[2-(13)C]Leu16 was measured by rotational resonance. As expected, the internuclear distances measured in [1-(13)C]Gly3-[2-(13)C]Ala4 and [1-(13)C]Gly3-[2-(13)C]Leu6 were consistent with alpha-helical structure in the N-terminus irrespective of environment. The internuclear distances measured in [1-(13)C]Leu13-[2-(13)C]Ala15, [2-(13)C]Leu13-[1-(13)C]Ala15, and [1-(13)C]Leu13-[2-(13)C]Leu16 revealed, via molecular modeling, some dependence upon environment for conformation in the region of the bend in helical structure induced by Pro14. A slightly larger interhelical angle between the N- and C-terminal helices was indicated for peptide in dry or hydrated gel state DTPC (139 degrees -145 degrees ) than in lyophilized powder (121 degrees -139 degrees ) or crystals (129 degrees ). The angle, however, is not as great as deduced for melittin in aligned bilayers of DTPC in the liquid-crystalline state (approximately 160 degrees ). The study illustrates the utility of rotational resonance in determining local structure within peptide-lipid complexes.

The properties of phosphatidylcholines (PCs) having a perdeuterated stearic acid, 18:0d35, in the sn-1 position and the fatty acid 18:0, 18:1 omega 9, 18:2 omega 6, 18:3 omega 3, 20:4 omega 6, 20:5 omega 3, or 22:6 omega 3 at the sn-2 position were investigated in a matrix of dioleoylphosphatidylethanolamine (DOPE) by 2H and 31P NMR spectroscopy. At a mole ratio of DOPE/PC = 5:1, the lipids form liquid crystalline lamellar phases below 40 degrees C and coexisting lamellar, inverse hexagonal (Hll), and cubic phases at higher temperatures. The sn-1 chain of the PCs in a DOPE matrix is appreciably more ordered than in pure PCs, corresponding to an increase in the hydrophobic bilayer thickness of approximately 1 A. Distearoylphosphatidylcholine in the DOPE matrix has a higher sn-1 chain order than the unsaturated PCs. We observed distinct differences in the lipid order of upper and lower sections of the hydrocarbon chains caused by changes of temperature, unsaturation, headgroups, and ethanol. Unsaturation lowers chain order, mostly in the lower third of the hydrocarbon chains. By contrast, the increase in chain order caused by the DOPE matrix and the decrease in order with increasing temperature have a constant magnitude for the upper two-thirds of the chain and are smaller for the lower third. Addition of 2 M ethanol reduced order parameters, in effect reversing the increase in chain order caused by the DOPE matrix.

Gramicidin A analogs, labeled with 13C in the backbone carbonyl groups and the C-2 indole carbons of the tryptophan-11 and tryptophan-13 residues, were synthesized using t-Boc-protected amino acids. The purified analogs were incorporated into phosphatidylcholine bilayers at a 1:15 molar ratio and macroscopically aligned between glass coverslips. The orientations of the labeled groups within the channel were investigated using solid-state NMR and the effect of a monovalent ion (Na+) on the orientation of these groups determined. The presence of sodium ions did not perturb the 13C spectra of the tryptophan carbonyl groups. These results contrast with earlier results in which the Leu-10, Leu-12, and Leu-14 carbonyl groups were found to be significantly affected by the presence of sodium ions and imply that the tryptophan carbonyl groups are not directly involved in ion binding. The channel form of gramicidin A has been demonstrated to be the right-handed form of the beta 6.3 helix: consequently, the tryptophan carbonyls would be directed away from the entrance to the channel and take part in internal hydrogen bonding, so that the presence of cations in the channel would have less effect than on the outer leucine residues. Sodium ions also had no effect on the C-2 indole resonance of the tryptophan side chains. However, a small change was observed in Trp-11 when the ether lipid, ditetradecylphosphatidylcholine, was substituted for the ester lipid, dimyristoylphosphatidylcholine, indicating some sensitivity of the gramicidin side chains to the surrounding lipid.

Solid-state 1H, 13C, 14N, and 31P NMR spectroscopy was used to study the effects of the bee venom peptide, melittin, on aligned multilayers of dimyristoyl-, dilauryl- and ditetradecyl-phosphatidylcholines above the gel to liquid-crystalline transition temperature, Tc. Both 31P spectra from the lipid headgroups and 1H resonances from the lipid acyl chain methylene groups indicate that the peptide does not affect the mosaic spread of the lipid molecules at lipid:peptide molar ratios of 10:1, or higher. None of the samples prepared above Tc showed any evidence of the formation of hexagonal or isotropic phases. Melittin-induced changes in the chemical shift anisotropy of the headgroup phosphate and the lipid carbonyl groups, and in the choline 14N quadrupole splittings, show that the peptide has effects on the headgroup order and on the molecular organization in the sections of the acyl chains nearest to the bilayer surface. The spin-lattice relaxation time for the lipid acyl chain methylene protons was found to increase and the rotating-frame longitudinal relaxation time to markedly decrease with the addition of melittin, suggesting that motions on the nanosecond time scale are restricted, whereas the slower, collective motions are enhanced in the presence of the peptide.

Three analogues of the helical ionophore gramicidin A have been synthesized with (13)C-labeled carbonyls ((13)C=O) incorporated at either Gly(2), Ala(3), or Val(7). A fourth compound incorporated (13)C at both the carbonyl and alpha-carbon of Gly(2) within the same molecule. These labels were studied using solid-state, proton-enhanced, (13)C nuclear magnetic resonance (NMR) in hydrated dispersions of dimyristoylphosphatidylcholine (DMPC)-gramicidin A. The dispersions were aligned on glass coverslips whose orientation to the magnetic field could be varied through 180 degrees . The orientation dependence of the NMR spectrum was used to obtain an accurate measurement of the (13)C chemical shift anisotropy (CSA), and in the case of the fourth compound, the (13)C-(13)C dipolar coupling constant. From the measured CSA and estimates of the orientation of the (13)C shielding tensor, we are able to determine the direction of the (13)C=O bonds and to compare these with the predictions of the various reported models for the configuration of gramicidin A in phospholipid bilayers. Our results are consistent with the left-handed pipi(6.3) (LD) single-stranded helix (Urry, D. W., J. T. Walker, and T. L. Trapane. 1982. J. Membr. Biol. 69:225-231). The right-handed pipi(6.3) (LD) single-stranded helix observed for gramicidin A in sodium dodecyl sulfate micelles (Arseniev, A. S., I. L. Barsukov, V. F. Bystrov, A. L. Loize, and Yu A. Ovchinnikov. 1985. FEBS (Fed. Eur. Biochem. Soc.) Lett. 186:168-174) yields a poorer fit to the data. However, the width of the carbonyl resonances suggests a distribution of molecular geometries possibly resulting from a spread in the helix pitch and handedness. Double-stranded helices and beta sheet structures are excluded. In dispersions in which the lipid is in the L(alpha) phase, the gramicidin A undergoes rapid reorientation about an axis which is centered on the normal to the plane of the coverslips. When the supporting lipid is in the L(beta') phase the helices are rigid on the timescale of (13)C-NMR. The configuration of gramicidin A is unaltered by L(alpha)-L(beta') phase transition of the bilayer lipid.

Solid-state nuclear magnetic resonance (NMR) measurements on 13C-labeled analogues of the ion channel-forming peptide, gramicidin A, have been used to directly determine the structure of this peptide in lipid membranes. Seven gramicidin analogues, each labeled in a single carbonyl group of gly2, L-ala3, D-leu4, L-val7, D-leu10, D-leu12, or D-leu14 were synthesized by the solid-phase method. These gramicidin analogues were incorporated into aligned multilayers of dimyristoylphosphatidylcholine, or diether lipid bearing 14- or 16-carbon chains, at a 1:15 peptide:lipid mole ratio. Proton-enhanced, 13C, solid-state spectra were obtained at several temperatures and over a range of sample orientations with respect to the spectrometer magnetic field to permit accurate measurement of the chemical shift anisotropies. The observed anisotropies indicate that all of the labeled carbonyl bonds are oriented almost parallel to the molecular long axis and perpendicular to the lipid bilayer plane. These orientations are consistent with gramicidin forming a beta 6.3 single-strand helix that is oriented parallel to the methylene chains of the lipid molecules. Comparison of the linewidths from labeled residues that are in the innermost turn of the helix (gly2, ala3, and D-leu4), in the center of the molecule (val7), and in the turn nearest the lipid bilayer surface (D-leu10, D-leu12, and D-leu14) suggests that although the peptide behaves largely as a rigid barrel, segments of the peptide close to the membrane surface possess greater motional freedom. At temperatures above the gel-to-liquid crystalline transition temperature (Tc) the gramicidin molecules rotate, with a less than millisecond correlation time, about the bilayer normal: several degrees below Tc they become immobile on the NMR timescale, without change in the channel conformation. In the L beta' phase the linewidths of the D-leu10, D-leu'2, and D-leu" resonances become equal to those of the other labeled sites, indicating reduced but equivalent motion for all of the peptide carbonyl groups.