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.     

Orientation of the valine-1 side chain of the gramicidin transmembrane channel and implications for channel functioning. A 2H NMR study.

The orientation of the valine-1 side chain of gramicidin was determined by solid-state 2H NMR using valine-1-deuterated (d8) gramicidin. The peptide was incorporated into DMPC bilayers that were oriented between glass plates. When the plates were oriented with their normal perpendicular to the magnetic field, four quadrupolar splittings were observed of 106, 68, 9.7, and 2.0 kHz. These resonances were assigned to C alpha D, C beta D, and the deuterons of each of the C gamma D3 methyl groups, respectively. The average orientation of the various C-D bonds was calculated with respect to the helix axis. The angle obtained for the C alpha-D resonance was consistent with a single-stranded beta 6.3-helical model for the backbone but not with double-helical models. The angles of the side chain were then fitted to a model for the right-handed beta 6.3-helix. Rotation of the valine-1 side chain yielded a set of torsion angles that matched the angles as determined from the 2H NMR measurements. The corresponding orientation of the valine-1 side chain (chi 1 = -5 degrees) was found to be quite unusual, but it explains well the importance of a branched side chain at position 1 for channel formation and stability. A van der Waals interaction between valine-1 of one monomer and alanine-5 of the other helps to stabilize the gramicidin dimer.     

Spatial structure of gramicidin A in organic solvents. 1H-NMR analysis of conformation heterogeneity in ethanol

Structural features of double helices formed by polypeptides with alternating L- and D-amino acid residues were analysed. It was found that the map of short distances (less than 4 A) between protons of the two backbones is unique for each double helix type and even its fragment implies unambiguously parameters of the helix (i.e. parallel or antiparallel, handedness, pitch of helix, relative shift of polypeptide chains). By analysis of two-dimensional 1H-NMR spectra (COSY, RELSY, HOHAHA, NOESY), proton resonances of [Val1]gramicidin A (GA) in the ethanol solution were assigned. The results obtained show that the solution contains five stable conformations of GA in comparable concentrations. Monomer of GA is in a random coil conformation. Specific maps of short interproton distances for the other four species (1-4) were obtained by means of two dimensional nuclear Overhauser effect spectroscopy. The maps as well as spin-spin couplings of the H-NC alpha-H protons and solvent accessibilities of the individual amide groups correspond to four types of double helices pi pi LD 5,6 with 5.6 residues per turn. The double helices are related to the Veatch species 1-4 of GA. Species 1 and 2 are left-handed parallel double helices increase increase pi pi LD 5,6 with different relative shift of polypeptide chains. Species 3 is a left-handed antiparallel double helix increase decrease pi pi LD 5,6 and species 4 is a right-handed parallel double helix increase increase LD 5,6. In the dimers helices are fixed by the maximum number (28) of interbackbone hydrogen bonds NH...O = C possible for these structures. Species 1, 3 and 4 have C2 symmetry axes. Relationship between gramicidin A spatial structures induced by various media is discussed.     

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.     

Effect of calcium on the dynamic behavior of sialylglycerolipids and phospholipids in mixed model membranes. A 2H and 31P NMR study

DTSL, a sialic acid bearing glyceroglycolipid, has been deuteriated at the C3 position of the sialic acid headgroup and at the C3 position of the glycerol backbone. The glycolipid was studied as a neat dispersion and in multilamellar dispersions of DMPC (at a concentration of 5-10 mol % relative to phospholipid), using 2H and 31P NMR. The quadrupolar splittings, delta v Q, of the headgroup deuterons were found to differ in the neat and mixed dispersion, suggesting different headgroup orientations in the two systems. In DTSL-DMPC liposomes, two quadrupolar splittings were observed, indicating that the axial and equatorial deuterons make different angles with respect to the axis of motional averaging. The splittings originating from the equatorial and axial deuterons were found to increase and decrease with increasing temperature, respectively, indicating a temperature-dependent change in average headgroup orientation. Longitudinal relaxation times, T1Z, were found to be short (3-6 ms). The field dependence of T1Z suggests that more than one motion governs relaxation. At 30.7 MHz a T1Z minimum was observed at approximately 40 degrees C. At 46.1 MHz the T1Z values were longer and increased with temperature, demonstrating that the dominant rigid-body motions of the headgroup at this field are in the rapid motional regime (greater than 10(8) s-1). DTSL labeled at the glycerol C3 position was studied in DMPC multilamellar dispersions. Whereas two quadrupolar splittings have been observed for other glycolipids labeled at this position, only a single delta nu Q was observed. This shows that the orientation of the C2-C3 segment of DTSL relative to the bilayer normal differs from that of other glycolipids.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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).     

Energy-minimized conformation of gramicidin-like channels. I. Infinitely long poly-(L,D)-alanine beta 6.3-helix.

The energy-minimized conformation of an infinitely long poly-(L,D)-alanine in single-stranded beta 6.3-helix was calculated by the molecular mechanics method. When energy minimization was started from a wide range of initial geometries, six optimized conformations were obtained and identified as the right- and left-handed counterparts of the beta 4.5-, beta 6.3-, and beta 8.2-helices. It was found that their conformation energies increase in this order, the beta 4.5-helix having the lowest energy. The backbone dihedral angles of the energy-minimized beta 6.3-helix were: phi L = -116 degrees (or -131 degrees), psi L = 122 degrees (or 111 degrees), phi D = 131 degrees (or 116 degrees), psi D = -111 degrees (or -122 degrees), omega L = 173 degrees (or 173 degrees), and omega D = -173 degrees (or -173 degrees) for the right-handed (or left-handed) helix. This helix was composed of 6.30 residues/turn with a pitch of 4.97 A. All the alpha-carbons of L- and D-configurations appeared on one common circular helix. Interestingly, small deviations (approximately 7 degrees) of the peptide bonds from the planar structure caused a considerable lowering of the conformation energy, and, at the same time, they produced more favorable fitting of the hydrogen bonds; the carbonyl oxygens and the nearest-neighbor alpha-hydrogens also took more favorable relative positions.     

On the helix sense of gramicidin A single channels.

In order to resolve whether gramicidin A channels are formed by right- or left-handed beta-helices, we synthesized an optically reversed (or mirror image) analogue of gramicidin A, called gramicidin A-, to test whether it forms channels that have the same handedness as channels formed by gramicidin M- (F. Heitz et al., Biophys. J. 40:87-89, 1982). In gramicidin M- the four tryptophan residues have been replaced with phenylalanine, and the circular dichroism (CD) spectrum therefore reflects almost exclusively contributions from the polypeptide backbone. The CD spectrum of gramicidin M- in dimyristoylphosphatidylcholine vesicles is consistent with a left-handed helical backbone folding motif (F. Heitz et al., Biophys. Chem. 24:149-160, 1986), and the CD spectra of gramicidins A and A- are essentially mirror images of each other. Based on hybrid channel experiments, gramicidin A- and M- channels are structurally equivalent, while gramicidin A and A- channels are nonequivalent, being of opposite helix sense. Gramicidin A- channels are therefore left-handed, and natural gramicidin A channels in phospholipid bilayers are right-handed beta 6.3-helical dimers.     

A conformational rearrangement in gramicidin A: from a double-stranded left-handed to a single-stranded right-handed helix.

A conformational transition is described for the polypeptide, gramicidin A, in which a dimer that forms a left-handed intertwined antiparallel helix is converted to a single-stranded amino terminus to amino terminus right-handed helix. The starting structure is determined here by solution NMR methods while reference is made to the well-established folding motif of gramicidin in a lipid bilayer for the ultimate conformation of this transition. Furthermore, an organic solvent system of benzene and ethanol in which gramicidin has a unique conformation is identified. This conformation is shown to be very similar to that derived from X-ray diffraction of crystals prepared from a similar solvent system.     

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.     

A model of orientational ordering in phosphatidylcholine bilayers based on conformational analysis of the glycerol backbone region.

Molecular and conformational ordering in aqueous multilamellar suspensions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) have been examined by deuterium nuclear magnetic resonance (2H NMR) in the liquid crystalline (L alpha) phase. Motionally averaged quadrupolar splittings vQ from six sites in the vicinity of the glycerol backbone have been analyzed by a molecular frame and order matrix approach in which the usual assumption of a freely-rotating molecule is not invoked. By assuming a relatively rigid glycerol backbone region, the six vQ values are found to be consistent with a conformation of the glycerol backbone that is almost identical to that of one of the two structures in crystalline DMPC dihydrate (Pearson, R. H., and I. Pascher, 1979, Nature (Lond.) 281: 499-501). The orientation of the most-ordered axis of the DMPC molecule is found to be tilted at an angle of 27 +/- 2 degrees with respect to the long axis of the sn-1 chain in its extended all trans conformation. The ordering of the most ordered molecular axis with respect to the bilayer normal is expressed by an order parameter of Szz approximately equal to 0.6 +/- 0.1, consistent with values in analogous thermotropic liquid crystals.     

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.     

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.     

The orientation of (-)-delta 9-tetrahydrocannabinol in DPPC bilayers as determined from solid-state 2H-NMR

The orientation of the motional axis of (-)-delta 9-tetrahydrocannabinol in dipalmitoylphosphatidylcholine model membrane was calculated from the 2H quadrupolar splittings (delta nu Q) of individual deuterons strategically located on the cannabinoid tricyclic component. The molecule assumes an orientation in which its long axis is nearly perpendicular to the phospholipid chains and its most ordered axis is almost in the plane of the aromatic ring. This 'awkward' cannabinoid orientation in the membrane presumably occurs in order to allow the phenolic hydroxyl group to direct itself towards the polar bilayer interface.     

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)     

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.     

Constant helical pitch of the gramicidin channel in phospholipid bilayers.

X-ray diffraction has been applied in measuring the helical pitch of the gramicidin channel in oriented bilayers of dilauroylphosphatidylcholine (DLPC) and dimyristoylphosphatidylcholine (DMPC) at a polypeptide concentration of 9.1 mol %. The diffraction data show the helical pitch of gramicidin to be 4.7 +/- 0.2 A in both gel and liquid-crystalline phase bilayers, with and without monovalent cations. In addition, the width of the reflection due to the pitch of the helical gramicidin channel is consistent with a five turn helix.     

Methylation of cytosine in the 5-position alters the structural and energetic properties of the supercoil-induced Z-helix and of B-Z junctions

The structural and energetic consequences of cytosine methylation in the 5-position on the supercoil-dependent B-Z equilibrium in alternating dC-dG sequences cloned into recombinant plasmids were investigated. The helical parameters determined with the band shift method for right-handed [10.7 base pairs (bp)/turn] and left-handed (12.8 bp/turn) 5MedC-dG inserts were different from the helical repeat values for unmethylated dC-dG inserts (10.5 bp/turn in the right-handed and 11.5 bp/turn in the left-handed form). We analyzed the thermodynamic parameters delta GBZ (free energy difference per base pair between right-handed and left-handed helix structure), delta Gjx (free energy for formation of one B-Z junction), and b (helix unwinding at a junction region) for varying lengths of dC-dG inserts by two-dimensional gel electrophoresis and application of a statistical mechanics model. A comparison of plasmids fully methylated in vitro with HhaI methylase and their unmethylated counterparts revealed that delta Gjx is not significantly changed by cytosine methylation. However, this base modification results in an approximate 3-fold decrease of delta GBZ and an approximate 2-fold decrease of the unwinding b at B-Z junction regions. Analysis of a pair of related plasmids, each containing two dC-dG blocks, revealed qualitatively different transition behaviors. When the two dC-dG blocks were separated by 95 bp of a mixed sequence, they underwent independent B to Z transitions with separate nucleation events and junction formations. When the two blocks were separated by only a 4 bp GATC sequence, only one nucleation event was necessary, and the Z-helix spread across the nonalternating GATC region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of 19-nor-10-keto-25-hydroxyvitamin D3 in cultured mammalian cells

The results of normal mode calculations on the beta 4.4, beta 6.3, beta 5.6, and beta 7.2 structures of gramicidin A are compared with infrared and Raman spectra of crystalline native, crystalline Cs+-bound, and vesicle-bound gramicidin A. The observed frequencies and frequency splittings are in good agreement with an assignment of beta 5.6, beta 7.2, and beta 6.3 structures, respectively, to the gramicidin A molecules in the above three systems.