Orientations of the tryptophan 9 and 11 side chains of the gramicidin channel based on deuterium nuclear magnetic resonance spectroscopy.

Deuterium nuclear magnetic resonance spectroscopy was used to investigate the orientations of the indole rings of Trp9 and Trp11 in specific indole-d5-labeled samples of gramicidin A incorporated into dimyristoyl phosphatidylcholine bilayers in the beta 6.3 channel conformation. The magnitudes and signs of the deuterium quadrupolar splittings were fit to the rings and assigned to specific ring bonds, using a full rotation search of the chi 1 and chi 2 angles of each Trp and a least-squares method. Unique assignments were obtained. The data and assignments are in close agreement with four sets of (chi 1, chi 2) angles for each Trp in which the indole N-H is oriented toward the membrane's exterior surface. (Four additional sets of (chi 1, chi 2) angles with the N-H's pointing toward the membrane interior are inconsistent with previous observations.) One of the sets of (chi 1, chi 2) angles for each Trp is consistent with the corresponding Trp orientation found by Arsen'ev et al. (1986. Biol. Membr. 3:1077-1104) for gramicidin in sodium dodecyl sulfate micelles. Together, the 1H and 2H nuclear magnetic resonance methods suggest that the Trp9 and Trp11 side chain orientations could be very similar in dimyristoyl phosphatidylcholine membranes and in sodium dodecyl sulfate micelles. The data for Trp11 could be fit using a static quadrupolar coupling constant of 180 kHz under the assumption that the ring is essentially immobile. By contrast, Trp9 could be fit only under the assumption that the quadrupolar splittings for ring 9 are reduced by approximately 14% due to motional averaging. Such a difference in motional averaging between rings 11 and 9 is also consistent with the 15N data of Hu et al. (1993. Biochemistry. 32:7035-7047).     

Deuterium NMR of Val1...(2-2H)Ala3...gramicidin A in oriented DMPC bilayers.

Deuterium NMR is used to study the selectively labeled Val1...(2-2H)Ala3...gramicidin A molecule to investigate the structure and dynamics of the C alpha-2H bond in the Ala3 residue of gramicidin. Val1...(2-2H)Ala3...gramicidin A is synthesized, purified, and characterized and then incorporated into oriented bilayers of dimyristoylphosphatidylcholine sandwiched between glass coverslips. Phosphorus NMR line shapes obtained from this sample are consistent with the presence of the bilayer phase and indicate that no nonbilayer phases are present in significant amounts. Deuterium NMR line shapes obtained from this sample indicate that the motional axis of the gramicidin Ala3 residue is parallel to the coverslip normal, that the distribution of motional axis orientations has a width of 2 degrees, and that only one major conformational and dynamical state of the Ala3 C alpha-2H bond is observed on the NMR time scale. Furthermore, the Ala3 C alpha-2H bond angle relative to the motional axis is 19-20 degrees if fast axial rotation is assumed to be the only motion present but is less than or equal to 19-20 degrees in the absence of such an assumption. This result indicates that various double-stranded, helical dimer models are very unlikely to represent the structure of gramicidin in the sample studied but that the single-stranded, beta 6.3 helical dimer models are consistent with the experimental data. However, a definitive distinction between the left-handed, single-stranded, beta 6.3 helical dimer model and the right-handed, single-stranded, beta 6.3 helical dimer model cannot be made on the basis of the experimental data obtained in this study.     

Modulating dipoles for structure-function correlations in the gramicidin A channel.

Dipoles of the tryptophan indole side chains have a direct impact on ion conductance in the gramicidin channel. Here, fluorination of the indoles (both 5- and 6-fluoro) is used to manipulate both the orientations and the magnitudes of the dipoles. The orientations and positions with respect to the channel axis were determined using (2)H solid state NMR of uniformly aligned lipid bilayer preparations. By exchange of the remaining four protons in the indole ring for deuterium, comparison could be made to d(5)-indole spectra that have previously been recorded for each of the four indoles of gramicidin A. After making the assignments which were aided by the observation of (19)F-(2)H dipolar interactions, we found that fluorination caused only minor changes in side chain conformation. With the high-resolution structural characterization of the fluorinated indoles in position 11, 13, and 15, the electrostatic interactions with a cation at the channel and bilayer center can be predicted and the influence of the modified dipoles on ion conductance estimated. The importance of the long-range electrostatic interaction was recently documented with the observation of alpha-helical dipoles oriented toward the bilayer center on the ion conductance pathway for the Streptomyces K(+) channel. We present direct measurements of the orientation of gramicidin channel F-Trp positions for use in analysis of dipole effects on channel permeation.     

Deuterium NMR of 2HCO-Val1...gramicidin A and 2HCO-Val1-D-Leu2...gramicidin A in oriented DMPC bilayers.

Deuterium NMR is used to study the structure and dynamics of the formyl C-2H bond in selectively deuterated gramicidin molecules. Specifically, the functionally different analogues 2HCO-Val1...gramicidin A and 2HCO-Val1-D-Leu2...gramicidin A are studied by 2H NMR so that any conformational or dynamical differences between the two analogues can be correlated with their difference in lifetime. These analogues are first synthesized, purified, and characterized and then incorporated into oriented bilayers of dimyristoylphosphatidylcholine sandwiched between glass coverslips. Phosphorous NMR line shapes obtained from these samples are consistent with the presence of the bilayer phase and indicate that the disorder exhibited by the lipid matrix is approximately of the same type and degree for both analogues. Deuterium NMR line shapes obtained from these samples indicate that the motional axis of the formyl group of gramicidin is parallel to the coverslip normal, that the distribution of motional axis orientations has a width of 7-9 degrees, and that a similar, major conformational and dynamical state exists for the formyl C-2H bond of both analogues. In this state, if the only motion present is fast axial rotation, then the experimentally derived angle between the formyl C-2H bond and the motional axis is consistent with the presence of a right-handed, single-stranded, beta 6.3 helical dimer but is not consistent with the presence of a left-handed, single-stranded, beta 6.3 helical dimer. However, if fast axial rotation is not the only motion present, then the left-handed, single-stranded, beta 6.3 helical dimer cannot be absolutely excluded as a possibility. Also, a second, minor conformational and dynamical state appears to be present in the spectrum of 2HCO-Val1-D-Leu2...gramicidin A but is not observed in the spectrum of 2HCO-Val1...gramicidin A. This minor conformational and dynamical state may reflect the presence of monomers, while the major conformational and dynamical state may reflect the presence of dimers.     

2H nuclear magnetic resonance of exchange-labeled gramicidin in an oriented lyotropic nematic phase

Lyotropic nematic liquid-crystalline phases, such as that formed by potassium laurate/decanol/KCl/water, are found to accept readily large amphiphilic solute molecules. Since these phases spontaneously orient in high magnetic fields, it becomes possible to obtain NMR spectra of biologically interesting solutes in an oriented axially symmetric environment. The amide hydrogens of the peptide backbone of gramicidin D (Dubos) were exchanged for deuterium, and the gramicidin was incorporated into a lyotropic nematic phase made with deuteriated buffer in place of water. 2H NMR spectra of oriented, exchange-labeled gramicidin were then obtained. The strong water signal from the deuteriated buffer was eliminated by using selective excitation and a polynomial subtraction procedure. The 2H NMR spectra at high temperature consist of twelve major quadrupolar doublets. The splittings observed are largely independent of temperature, suggesting a highly rigid backbone structure. Two of the doublets, which are chemically shifted relative to the others, show stronger temperature dependence. These two probably arise from the exchangeable amino hydrogens on the tryptophan indole moieties of the peptide. While we cannot yet assign all of the doublets, the spectra and nuclear magnetic relaxation data are consistent with a rigid slightly distorted beta LD6.3 helix undergoing axially symmetric reorientation about the director of the liquid-crystalline phase. The correlation time for the axially symmetric reorientation is determined by relaxation measurements to be about 10(-7) s.     

Neighboring aliphatic/aromatic side chain interactions between residues 9 and 10 in gramicidin channels

The interactions between an aliphatic or phenyl side chain and an indole ring in a phospholipid environment were investigated by synthesizing and characterizing gramicidins in which Trp(9) was ring-labeled and D-Leu(10) was replaced by D-Val, D-Ala, or D-Phe. All three analogues form conducting channels, with conductances that are lower than that of gramicidin A (gA) channels. The channel lifetimes vary by less than 50% from that of gA channels. Circular dichroism spectra and size-exclusion chromatography show that the conformation of each analogue in dimyristoylphosphatidylcholine (DMPC) vesicles is similar to the right-handed beta(6.3)-helical conformation that is observed for gA. (2)H NMR spectra of oriented samples in DMPC show large changes for the Trp(9) ring when residue 10 is modified, suggesting a steric interaction between D-Leu(10) and Trp(9), in agreement with previous acylation studies (R. E. Koeppe II et al. (1995) Biochemistry 34, 9299-9307). The outer quadrupolar splitting for Trp(9) is unchanged with D-Phe(10), at approximately 153 kHz, but increases by approximately 25 kHz with D-Val(10) and decreases by approximately 10 kHz with D-Ala(10). With D-Ala(10) or D-Val(10), the outer resonance splits into two in a temperature-dependent manner. The NMR spectra indicate that the side chain torsion angles chi1 and chi2 for Trp(9) change when residue 10 is substituted. The changes in chi1 are small, in all cases less than 10 degrees, as is Deltachi2 when D-Ala(10) is introduced, but with D-Val(10) and D-Phe(10) Deltachi2 is at least 25 degrees. We conclude that D-Leu(10) helps to stabilize an optimal orientation of Trp(9) in gA channels in lipid bilayers and that changes in Trp orientation alter channel conductance and lifetime without affecting the basic channel fold.     

Orientation of gramicidin A transmembrane channel. Infrared dichroism study of gramicidin in vesicles.

Polarized infrared spectroscopy has been used to investigate the orientation of gramicidin A incorporated in dimyristoylphosphatidylcholine liposomes. Dichroism measurements of the major lipid (C = O ester, PO2-, CH2) and peptide (amide A, I, II) bands were performed on liposomes (with or without gramicidin) oriented by air-drying. The mean orientation of the lipid groups and of the pi LD helix chain in the gramicidin has been determined. It can be inferred from infrared frequencies of gramicidin that the dominant conformation of the peptide in liposomes cannot be identified to the antiparallel double-helical dimer found in organic solution. No shift in lipid frequencies was observed upon incorporation of gramicidin in the liposomes. However, a slight reorganization of the lipid hydrocarbon chains which become oriented more closely to the normal to the bilayer is evidenced by a change in the dichroism of the CH2 vibrations. The infrared dichroism results of gramicidin imply a perpendicular orientation of the gramicidin transmembrane channel with the pi LD helix axis at less than 15 degrees with respect to the normal to the bilayer.     

2H nuclear magnetic resonance of the gramicidin A backbone in a phospholipid bilayer.

Solid-state 2H NMR spectroscopy has been employed to study the channel conformation of gramicidin A (GA) in unoriented 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC) multilayers. Quadrupolar echo spectra were obtained at 44 degrees C and 53 degrees C, from gramicidin A labels in which the proton attached to the alpha carbon of residue 3, 4, 5, 10, 12, or 14 was replaced with deuterium. Because of the nearly axially symmetric electric field gradient tensor, the quadrupolar splittings obtained from an unoriented multilamellar dispersion of DMPC and singly labeled GA directly yield unambiguous orientational constraints on the C-2H bonds. The average of the ratios of the quadrupolar splittings of the left-handed amino acids to those of the right-handed amino acids, (delta vQL/delta vQD), is expected to be 0.97 +/- 0.04 for a relaxed right-handed beta 6.3LD helix, while a ratio of 0.904 +/- 0.003 is expected for a left-handed beta LD6.3 helix. Since we have experimentally determined this ratio to be 1.01 +/- 0.04, we conclude that that the helix sense of the channel conformation of GA is right-handed. Assuming that the dominant motions are fast axial diffusion of the gramicidin molecule and reorientation of the diffusion axis with respect to the local bilayer normal, then the theoretical splittings may all be scaled down by a constant motional narrowing factor. In this case, a relaxed right-handed beta LD6.3 helix, whose axis of motional averaging is roughly along the presumed helix axis, gave the best fit to experimental results. The reasonably uniform correspondence between the splittings predicted by the relaxed right-handed beta LD6.3 helix and the observed splittings, for labels from both the inner and outer turn of GA, did not reflect a peptide backbone flexibility gradient, since an outer turn (i.e., the turn of the helix closest to the interface with water) with greater flexibility would show additional motional narrowing for labels located there.     

13C solid-state NMR of gramicidin A in a lipid membrane.

The natural-abundance 13C NMR spectrum of gramicidin A in a lipid membrane was acquired under magic-angle spinning conditions. With fast sample spinning (15 kHz) at approximately 65 degrees C the peaks from several of the aliphatic, beta-, alpha-, aromatic, and carbonyl carbons in the peptide could be resolved. The resolution in the 13C spectrum was superior that observed with 1H NMR under similar conditions. The 13C linewidths were in the range 30-100 Hz, except for the alpha- and beta-carbons, the widths of which were approximately 350 Hz. The beta-sheet-like local structure of gramicidin A was observed as an upfield shift of the gramicidin alpha and carbonyl resonances. Under slow sample spinning (500 Hz), the intensity of the spinning sidebands from 13C in the backbone carbonyls was used to determine the residual chemical shift tensor. As expected, the elements of the residual chemical shift tensor were consistent with the single-stranded, right-handed beta6.3 helix structure proposed for gramicidin A in lipid membranes.     

The orientation effect of gramicidin A on bicelles and Eu3+ -doped bicelles as studied by solid-state NMR and FT-IR spectroscopy

We have explored the effect of gramicidin A (gA) on bicelle (Bic) orientation in the absence and presence of Eu(3+) by (31)P and (2)H NMR at different DMPC/gA ratios. FT-IR spectroscopy was used to assess the lipid chain ordering and verify the transmembrane peptide conformation. Our results show a time-dependent flipping of the bilayer normal alignment at high temperatures and high proportion of gA. The results are explained by both the diamagnetic susceptibility anisotropy of the beta(6.3) helical peptides and viscosity of the lipid mixture. The concentration effect of gramicidin on Bic/Eu(3+) is compared to that on Eu(3+)-doped DMPC liposomes. The Bic/Eu(3+) system is no longer oriented in the presence of gA and adopts a vesicular morphology while the peptide incorporation induces the formation of ellipsoidal DMPC/Eu(3+) assemblies aligned with their normal parallel to the magnetic field. The difference is explained in terms of lipid chain disorder and size of the bilayers.     

Sodium ion binding in the gramicidin A channel. Solid-state NMR studies of the tryptophan residues.

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.     

Polarization-modulated FTIR spectroscopy of lipid/gramicidin monolayers at the air/water interface

Monolayers of gramicidin A, pure and in mixtures with dimyristoylphosphatidylcholine (DMPC), were studied in situ at the air/H2O and air/D2O interfaces by polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). Simulations of the entire set of amide I absorption modes were also performed, using complete parameter sets for different conformations based on published normal mode calculations. The structure of gramicidin A in the DMPC monolayer could clearly be assigned to a beta6.3 helix. Quantitative analysis of the amide I bands revealed that film pressures of up to 25-30 mN/m the helix tilt angle from the vertical in the pure gramicidin A layer exceeded 60 degrees. A marked dependence of the peptide orientation on the applied surface pressure was observed for the mixed lipid-peptide monolayers. At low pressure the helix lay flat on the surface, whereas at high pressures the helix was oriented almost parallel to the surface normal.     

Determination of the structure of a membrane-incorporated ion channel. Solid-state nuclear magnetic resonance studies of gramicidin A.

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.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phase equilibria and molecular packing in the N,N-dimethyldodecylamine oxide/gramicidin D/water system studied by 2H nuclear magnetic resonance spectroscopy.

A partial phase diagram of the system N,N-dimethyldodecylamine oxide (DDAO)/water/gramicidin D was determined by 2H-NMR. Both 2H2O and perdeuterated DDAO (DDAO-d31) were studied by solid state NMR techniques. Addition of gramicidin D to the micellar (L1), normal hexagonal (HI) and cubic (I) phases of DDAO induces phase separations, giving two-phase regions, which all contain a lamellar (L alpha) phase. The L alpha phase containing gramicidin is characterized by larger order parameters for DDAO-d31 compared with the corresponding order parameters in the L alpha and HI phases of DDAO-d31/H2O. The L alpha phase may stay in equilibrium with any other phase in the phase diagram. The DDAO exchange between the coexisting phases is slow on the NMR timescale, which is why the recorded NMR spectrum consists of superimposed spectra from the different phases occurring in the sample. Gramicidin D can be solubilized in appreciable quantities only in the lamellar phase of DDAO-d31. Increasing amounts of gramicidin in the liquid crystalline phases result in a continuous increase in the molecular ordering up to about 5 mol% gramicidin, where a plateau is reached. This is consistent with a recent theoretical model describing the influence on the ordering of lipids by a membrane protein with larger hydrophobic thickness than the lipid bilayer. The solvent used for dissolving gramicidin at the incorporation of the peptide in the lipid aggregates has no effect on the 2H-NMR lineshapes of DDAO-d31. It is concluded that gramicidin is solubilized in the L alpha phase and that it always adopts the channel conformation independent of a particular solvent. The channel conformation is also supported by CD studies. In some of the samples, macroscopic orientation of the lipid aggregates is observed. It is concluded that DDAO-d31 in the binary system favors an orientation with the long axis of the hydrocarbon chain perpendicular to the magnetic field, whereas when gramicidin D is present the hydrocarbon chain orients parallel to the magnetic field. This is explained by the fact that gramicidin aligns with its helical axis parallel to the magnetic field, thereby forcing also the DDAO-d31 molecules to obtain such an orientation.     

Structure determination of the cyclohexene ring of retinal in bacteriorhodopsin by solid-state deuterium NMR.

The orientation and conformation of retinal within bacteriorhodopsin of the purple membrane of Halobacterium halobium was established by solid-state deuterium NMR spectroscopy, through the determination of individual chemical bond vectors. The chromophore ([2,4,4,16,16,17,17,17,18,18-2H11]retinal) was specifically deuterium-labeled on the cyclohexene ring and incorporated into the protein. A uniaxially oriented sample of purple membrane patches was prepared and measured at a series of inclinations relative to the spectrometer field. 31P NMR was used to characterize the mosaic spread of the oriented sample, and computer simulations were applied in the analysis of the 2H NMR and 31P NMR spectral line shapes. From the deuterium quadrupole splittings, the specific orientations of the three labeled methyl groups on the cyclohexene ring could be calculated. The two adjacent methyl groups (on C1) of the retinal were found to lie approximately horizontal in the membrane and make respective angles of 94 degrees +/- 2 degrees and 75 degrees +/- 2 degrees with the membrane normal. The third group (on C5) points toward the cytoplasmic side with an angle of 46 degrees +/- 3 degrees. These intramolecular constraints indicate that the cyclohexene ring lies approximately perpendicular to the membrane surface and that it has a (6S)-trans conformation. From the estimated angle of the tilt of the chomophore long axis, it is concluded that the polyene chain is slightly curved downward to the extracellular side of the membrane.     

Side-chain structure and dynamics at the lipid-protein interface: Val1 of the gramicidin A channel.

High resolution dynamics and structural information has been resolved from 2H solid-state NMR spectra of the Val-1 side-chain of the gramicidin channel in a lipid bilayer. Both powder pattern lineshapes and spectra from uniformly aligned samples of gramicidin in lipid bilayers have been analyzed to achieve a fully consistant interpretation of the data. Torsional motions about the C alpha C beta axis (chi 1) are shown to be three-state jumps in which the occupancy of the states is given by the ratio, 75:15:10 for the chi 1 angles of 184 degrees:304 degrees:64 degrees. The dominant conformer is also the most common conformation observed for valines in well defined protein structures. The distribution of conformational substates that represents the chi 1 dynamics appears to be largely independent of the lipid phase transition and the hydration of the sample. However, there is evidence that the residence time between jumps is dependent on the lipid phase transition. Although this time is shown to be approximately 1 microseconds below the phase transition temperature, it is in the fast exchange limit above the transition temperature.     

The structure of an integral membrane peptide: a deuterium NMR study of gramicidin.

Solid state deuterium NMR was employed on oriented multilamellar dispersions consisting of 1,2-dilauryl-sn-glycero-3-phosphatidylcholine and deuterium (2H) exchange-labeled gramicidin D, at a lipid to protein molar ratio (L/P) of 15:1, in order to study the dynamic structure of the channel conformation of gramicidin in a liquid crystalline phase. The corresponding spectra were used to discriminate between several structural models for the channel structure of gramicidin (based on the left- and right-handed beta 6.3 LD helix) and other models based on a structure obtained from high resolution NMR. The oriented spectrum is complicated by the fact that many of the doublets, corresponding to the 20 exchangeable sites, partially overlap. Furthermore, the asymmetry parameter, eta, of the electric field gradient tensor of the amide deuterons is large (approximately 0.2) and many of the amide groups are involved in hydrogen bonding, which is known to affect the quadrupole coupling constant. In order to account for these complications in simulating the spectra in the fast motional regime, an ab initio program called Gaussian 90 was employed, which permitted us to calculate, by quantum mechanical means, the complete electric field gradient tensor for each residue in gramicidin (using two structural models). Our results indicated that the left-handed helical models were inconsistent with our observed spectra, whereas a model based on the high-resolution structure derived by Arseniev and coworkers, but relaxed by a simple energy minimization procedure, was consistent with our observed spectra. The molecular order parameter was then estimated from the motional narrowing assuming the relaxed (right-handed) Arseniev structure. Our resultant order parameter of SZZ = 0.91 translates into an rms angle of 14 degrees, formed by the helix axis and the local bilayer normal. The strong resemblance between our spectra (and also those reported for gramicidin in 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) multilayers) and the spectra of the same peptide incorporated in a lyotropic nematic phase, suggests that the lyotropic nematic phase simulates the local environment of the lipid bilayer.     

1H-NMR study of gramicidin A transmembrane ion channel. Head-to-head right-handed, single-stranded helices

The structure of [Val1]gramicidin A incorporated into sodium dodecyl-d25 sulphate micelles has been studied by two-dimensional proton NMR spectroscopy. Analysis of nuclear Overhauser effects, spin-spin couplings and solvent accessibility of NH groups show that the conformation of the Na+ complex of gramicidin A in detergent micelles, which in many ways mimic the phospholipid bilayer of biomembranes, is an N-terminal to N-terminal (head-to-head) dimer (Formula: see text) formed by two right-handed, single-stranded beta 6.3 helices with 6.3 residues per turn, differing from Urry's structure by handedness of the helices.     

The conformation of gramicidin A in dimethylsulphoxide solution. A full analysis of the one- and two-dimensional 1H, 13C, and 15N nuclear-magnetic-resonance spectra

The combined application of one- and two-dimensional high-field NMR techniques has led to the first assignment of the 1H, 13C, and 15N spectra of the pentadecapeptide gramicidin A in dimethylsulphoxide solution. The 62.9-MHz and 100.6-MHz 13C spin-lattice relaxation times and 13C-[1H] NOE factors for the backbone alpha carbons have been analysed in the 'model-free' approach to give a single correlation time (tau m) for isotropic overall molecular motion and an order parameter and internal correlation time for each C alpha H group in the backbone. The relatively high and constant values for the order parameter along the backbone indicate a degree of ordering of the structure, while the internal correlation times show that internal motions are progressively more rapid towards the N terminus. The average values of the vicinal HNC alpha H couplings are 7.4 Hz and 8.4 Hz respectively for the alternate L- and D-amino acid residues. The values are not consistent with either a ribbon conformation for the backbone or a right-handed beta 6.3 helix; they are consistent with the model proposed by Glickson et al. [Glickson, J. D., Mayers, D. F., Settine, J. M. & Urry, D. W. (1972) Biochemistry 11, 477-486] in which there is a rapid conformational order in equilibrium disorder equilibrium, the ordered structure being the left-handed beta 6.3 helix and the disordered state having local random-coil character.     

Solid-state 15N NMR of oriented lipid bilayer bound gramicidin A'

Highly oriented samples of lipid and gramicidin A' (8:1 molar ratio) have been prepared with the samples extensively hydrated (approximately 70% water v/w). These preparations have been shown to be completely in a bilayer phase with a transition temperature of 28 degrees C, and evidence is presented indicating that the gramicidin is in the channel conformation. An estimate of the disorder in the alignment of the bilayers parallel with the glass plates used to align the bilayers can be made from the asymmetry of the nuclear magnetic resonances (NMR). Such an analysis indicates a maximal range of disorder of +/- 3 degrees. Uniformly 15N-labeled gramicidin has been biosynthesized by Bacillus brevis grown in a media containing 15N-labeled Escherichia coli cells as the only nitrogen source. When prepared with labeled gramicidin, the oriented samples result in high-resolution 15N NMR spectra showing 12 resonances for the 20 nitrogen sites of the polypeptide. The frequency of the three major multiple resonance peaks has been interpreted to yield the approximate orientation of the N-H bonds in the peptide linkages with respect to the magnetic field. These bond orientations are only partially consistent with the extant structural models of gramicidin.