Solvent history dependence of gramicidin A conformations in hydrated lipid bilayers.

Reconstituted lipid bilayers of dimyristoylphosphatidylcholine (DMPC) and gramicidin A' have been prepared by cosolubilizing gramicidin and DMPC in one of three organic solvent systems followed by vacuum drying and hydration. The conformational state of gramicidin as characterized by 23Na NMR, circular dichroism, and solid state 15N NMR is dependent upon the cosolubilizing solvent system. In particular, two conformational states are described; a state in which Na+ has minimal interactions with the polypeptide, referred to as a nonchannel state, and a state in which Na+ interacts very strongly with the polypeptide, referred to as the channel state. Both of these conformations are intimately associated with the hydrophobic core of the lipid bilayer. Furthermore, both of these states are stable in the bilayer at neutral pH and at a temperature above the bilayer phase transition temperature. These results with gramicidin suggest that the conformation of membrane proteins may be dictated by the conformation before membrane insertion and may be dependent upon the mechanism by which the insertion is accomplished.     

Importance of hydration for gramicidin-induced hexagonal HII phase formation in dioleoylphosphatidylcholine model membranes

The macroscopic organization, lipid head group conformation, and structural and dynamic properties of 2H2O were investigated in dioleoylphosphatidylcholine (DOPC) model systems of varying gramicidin and 2H2O (or H2O) content by means of small-angle X-ray diffraction and 31P and 2H NMR. At low stages of hydration, N less than 6 (N = 2H2O/DOPC molar ratio), a single lamellar phase is observed in which the gramicidin molecules become preferentially hydrated upon increasing N. For 6 less than N less than 12 phase separation occurs between a gramicidin-poor and a gramicidin-rich lamellar phase. This latter phase is characterized by a smaller repeat distance and decreased DOPC head group order. For N greater than 12, the gramicidin-rich lamellar phase converts to a hexagonal HII phase. Thus, hydration of gramicidin is a prerequisite for HII phase formation in the DOPC/gramicidin system. The HII phase is very rich in gramicidin and 2H2O (gramicidin:DOPC:H2O = 1:1.1:0.9 w/w/w). A model is proposed in which self-assembly of hydrated gramicidin molecules into domains of a specific structure plays a determinant role in the formation of the HII phase by gramicidin.     

Gramicidin-induced hexagonal HII phase formation in erythrocyte membranes

Using 31P nuclear magnetic resonance (NMR), small-angle X-ray scattering (SAXS), and freeze-fracture electron microscopic (FFEM) techniques, it is shown that gramicidin induces a hexagonal HII phase not only in liposomes prepared from total lipids extracted from human erythrocytes but also in isolated human erythrocyte membranes (white ghosts). A 37 degrees C, HII phase formation is detected at a gramicidin to phospholipid molar ratio exceeding 1:80. At a molar ratio of 1:5, about 30% of the phospholipid is organized in the HII phase. The gramicidin-induced HII phase exhibits a very small 31P chemical shift anisotropy [(CSA) approximately 10 +/- 1 ppm], indicating decreased head-group order, and it displays a temperature-dependent increase in tube diameter from 60.2 A at 4 degrees C to 64.2 A at 37 degrees C in ghosts and from 62.8 to 69.4 A at 37 degrees C in total lipid extracts, both in the presence of 1 mol of gramicidin/10 mol of phospholipid. This anomalous temperature-dependent behavior is probably due to the presence of cholesterol. 31P NMR data indicate that the HII phase formation by gramicidin is temperature dependent and show the gradual disappearance of the HII phase at low temperatures (less than 20 degrees C), resulting in a bilayer type of 31P NMR line shape at 4 degrees C, whereas SAXS and FFEM data suggest equal amounts of HII phases at all temperatures. This apparent discrepancy is probably the result of a decrease in the rate of lateral diffusion of the membrane phospholipids which leads to incomplete averaging of the 31P CSA in the HII phase.(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.     

Novel properties of cholesterol-dioleoylphosphatidylcholine mixtures

We have studied the properties of mixtures of cholesterol with dioleoylphosphatidylcholine (DOPC), and with several other phospholipids, including 1-stearoyl-2-oleoylphosphatidylcholine (SOPC) and dioleoleoylphosphatidylserine (DOPS), as a function of cholesterol molar fraction and of temperature. Mixtures of DOPC with a cholesterol molar fraction of 0.4 or greater display polymorphic behavior. This polymorphism includes the formation of structures that give rise to isotropic peaks in 31P NMR at cholesterol molar fractions between 0.4 and 0.6, dependent on the thermal history of the sample. Cryo-electron microscopy studies demonstrate the formation of small globular aggregates that would contribute to a narrowing of the 31P NMR powder pattern.At molar fraction cholesterol 0.6 and higher and at temperatures above 70 degrees C, the mixtures with DOPC convert to the hexagonal phase. Lipid polymorphism is accompanied by the phase separation of cholesterol crystals in the anhydrous form and/or the monohydrate form. The crystals that are formed have substantially altered kinetics of hydration and dehydration, compared with both pure cholesterol monohydrate crystals and with crystals formed in the presence of the other phospholipids that do not form the hexagonal phase in the presence of cholesterol. This fact demonstrates that these cholesterol crystals are in intimate contact with the DOPC phospholipid and are not present as morphologically separate structures.     

Effect of graded hydration on the dynamics of an ion channel peptide: a fluorescence approach

Water plays an important role in determining the folding, structure, dynamics, and, in turn, the function of proteins. We have utilized a combination of fluorescence approaches such as the wavelength-selective fluorescence approach to monitor the effect of varying degrees of hydration on the organization and dynamics of the functionally important tryptophan residues of gramicidin in reverse micelles formed by sodium bis(2-ethylhexyl) sulfosuccinate. Our results show that tryptophans in gramicidin, present in the single-stranded beta6.3 conformation, experience slow solvent relaxation giving rise to red-edge excitation shift (REES). In addition, changes in fluorescence polarization with increasing excitation or emission wavelength reinforce that the gramicidin tryptophans are localized in motionally restricted regions of the reverse micelle. Interestingly, the extent of REES is found to be independent of the [water]/[surfactant] molar ratio (w(o)). We attribute this to heterogeneity in gramicidin tryptophan localization. Fluorescence intensity and mean fluorescence lifetime of the gramicidin tryptophans show significant reductions with increasing w(o) indicating sensitivity to increased polarity. Since the dynamics of hydration is related to folding, structure, and eventually function of proteins, we conclude that REES could prove to be a potentially sensitive tool to explore the dynamics of proteins under conditions of changing hydration.     

Novel properties of cholesterol-dioleoylphosphatidylcholine mixtures

We have studied the properties of mixtures of cholesterol with dioleoylphosphatidylcholine (DOPC), and with several other phospholipids, including 1-stearoyl-2-oleoylphosphatidylcholine (SOPC) and dioleoleoylphosphatidylserine (DOPS), as a function of cholesterol molar fraction and of temperature. Mixtures of DOPC with a cholesterol molar fraction of 0.4 or greater display polymorphic behavior. This polymorphism includes the formation of structures that give rise to isotropic peaks in ³¹P NMR at cholesterol molar fractions between 0.4 and 0.6, dependent on the thermal history of the sample. Cryo-electron microscopy studies demonstrate the formation of small globular aggregates that would contribute to a narrowing of the ³¹P NMR powder pattern.At molar fraction cholesterol 0.6 and higher and at temperatures above 70 °C, the mixtures with DOPC convert to the hexagonal phase. Lipid polymorphism is accompanied by the phase separation of cholesterol crystals in the anhydrous form and/or the monohydrate form. The crystals that are formed have substantially altered kinetics of hydration and dehydration, compared with both pure cholesterol monohydrate crystals and with crystals formed in the presence of the other phospholipids that do not form the hexagonal phase in the presence of cholesterol. This fact demonstrates that these cholesterol crystals are in intimate contact with the DOPC phospholipid and are not present as morphologically separate structures.     

Gramicidin-induced hexagonal HII phase formation in negatively charged phospholipids and the effect of N- and C-terminal modification of gramicidin on its interaction with zwitterionic phospholipids

The effect of gramicidin on macroscopic structure of the negatively charged membrane phospholipids cardiolipin, dioleoylphosphatidylglycerol and dioleoylphosphatidylserine in aqueous dispersions was investigated and compared with the effect of gramicidin on dioleoylphosphatidylcholine. It was shown by small-angle X-ray diffraction, 31P nuclear magnetic resonance and freeze-fracture electron microscopy that in all these lipid systems gramicidin is able to induce the formation of a hexagonal HII phase. 31P-NMR measurements indicated that the extent of HII phase formation in the various lipids ranged from about 40% to 60% upon gramicidin incorporation in a molar ratio of peptide to lipid of 1 : 10. Next, the following charged analogues of gramicidin were prepared: desformylgramicidin, N-succinylgramicidin and O-succinylgramicidin. The synthesis was verified with 13C-NMR and the effect of these analogues on lipid structure was investigated. It was shown that, as with gramicidin itself, the analogues induce HII phase formation in dioleoylphosphatidylcholine, lower and broaden the bilayer-to-HII phase transition in dielaidoylphosphatidylethanolamine and form lamellar structures upon codispersion with palmitoyllysophosphatidylcholine. Differential scanning calorimetry measurements indicated that, again like gramicidin, in phosphatidylethanolamine the energy content of the gel-to-liquid-crystalline phase transition is not affected by incorporation of the analogues, whereas in phosphatidylcholine a reduction of the transition enthalpy is found. These observations were explained in terms of a similar tendency to self-associate for gramicidin and its charged analogues. The results are discussed in the light of the various factors which have been suggested to be of importance for the modulation of lipid structure by gramicidin.     

Gramicidin A induced fusion of large unilamellar dioleoylphosphatidylcholine vesicles and its relation to the induction of type II nonbilayer structures.

The fusogenic properties of gramicidin were investigated by using large unilamellar dioleoylphosphatidylcholine vesicles. It is shown that gramicidin induces aggregation and fusion of these vesicles at peptide to lipid molar ratios exceeding 1/100. Both intervesicle lipid mixing and mixing of aqueous contents were demonstrated. Furthermore, increased static and dynamic light scattering and a broadening of 31P NMR signals occurred concomitant with lipid mixing. Freeze-fracture electron microscopy revealed a moderate vesicle size increase. Lipid mixing is paralleled by changes in membrane permeability: small solutes like carboxyfluorescein and smaller dextrans, FD-4(Mr approximately 4000), rapidly (1-2 min) leak out of the vesicles. However, larger molecules like FD-10 and FD-17 (Mr approximately 9400 and 17,200) are retained in the vesicles for greater than 10 min after addition of gramicidin, thereby making detection of contents mixing during lipid mixing possible. At low lipid concentrations (5 microM), lipid mixing and leakage are time resolved: leakage of CF shows a lag phase of 1-3 min, whereas lipid mixing is immediate and almost reaches completion during this lag phase. It is therefore concluded that leakage, just as contents mixing, occurs subsequent to aggregation and lipid mixing. Although addition of gramicidin at a peptide/lipid molar ratio exceeding 1/50 eventually leads to hexagonal HII phase formation and a loss of vesicle contents, it is concluded that leakage during fusion (1-2 min) is not the result of HII phase formation but is due to local changes in lipid structure caused by precursors of this phase. By making use of gramicidin derivatives and different solvent conformations, it is shown that there is a close parallel between the ability of the peptide to induce the HII phase and its ability to induce intervesicle lipid mixing and leakage. It is suggested that gramicidin-induced fusion and HII phase formation share common intermediates.     

Conformation of gramicidin in relation to its ability to form bilayers with lysophosphatidylcholine

The ability of gramicidin to induce bilayer formation in lysophosphatidylcholine (LPC) systems was investigated as a function of the conformation of the peptide. The conformation was varied by using different solvents to cosolubilize gramicidin and lipid. Using circular dichroism (CD), it was found that when codissolved in trifluoroethanol (TFE), after drying and subsequent hydration, gramicidin is mainly present in the single-stranded beta 6.3-helical configuration, whereas when using chloroform/methanol or ethanol as the solvent, it is proposed that the dominant conformation of gramicidin in the membrane is that of the double-stranded antiparallel dimer. Employing 31P NMR, the stoichiometry for bilayer formation was found to be 6 to 7 lipid molecules per gramicidin monomer, when samples were prepared from TFE, whereas a stoichiometry of 4 was found when chloroform/methanol or ethanol was the solvent. Upon heating the latter samples, a conversion was observed in the CD pattern toward that indicative of the beta 6.3-helical configuration. This change was accompanied by an increase in the extent of bilayer formation. Next, it was investigated whether the conformation of gramicidin and its ability to induce bilayer formation were dependent on the lipid acyl chain length. CD measurements of samples prepared from TFE indicated that gramicidin, independent of acyl chain length, was present in the beta 6.3-helical configuration but the intensity of the ellipticities at 218 nm increased with the length of the acyl chain. The extent of bilayer formation in these samples was found to be largely chain length independent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phase separation and hexagonal HII phase formation by gramicidins A, B and C in dioleoylphosphatidylcholine model membranes. A study on the role of the tryptophan residues

The role of the tryptophan-residues in gramicidin-induced HII phase formation was investigated in dioleoylphosphatidylcholine (DOPC) model membranes. 31P-NMR and small angle X-ray diffraction measurements showed, that gramicidin A and C (in which tryptophan-11 is replaced by tyrosine) induce a similar extent of HII phase formation, whereas for gramicidin B and synthetic analogs in which one tryptophan, either at position 9 or 11 is replaced by phenylalanine, a dramatic decrease of the HII phase inducing activity can be observed. Modification of all four tryptophans by means of formylation of the indole NH group leads to a complete block of HII phase formation. Sucrose density centrifugation experiments on the various peptide/lipid samples showed a quantitative incorporation of the peptide into the lipid. For all samples in a 1/10 molar ratio of peptide to lipid distinct bands were found, indicative of a phase separation. For the gramicidin A'/DOPC mixture these bands were analyzed and the macroscopic organization was determined by 31P-NMR and small-angle X-ray diffraction. The results demonstrate that a quantitative phase separation had occurred between a lamellar phase with a gramicidin/lipid ratio of 1/15 and a hexagonal HII phase, which is highly enriched in gramicidin. A study on the hydration properties of tryptophan-N-formylated gramicidin in mixtures with DOPC showed that this analog has a similar dehydrating effect on the lipid headgroup as the unmodified gramicidin. In addition both the hydration study and sucrose density centrifugation experiments showed that, like gramicidin also its analogs have a tendency to aggregate, but with differences in aggregation behaviour which seemed related to their HII phase inducing activity. It is proposed that the main driving force for HII phase formation is the tendency of gramicidin molecules to self-associate and organize into tubular structures such as found in the HII phase and that whether gramicidin (analogs) form these or other types of aggregates depends on their tertiary structure, which is determined by intra- as well as intermolecular aromatic-aromatic stacking interactions.     

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.     

NMR study of the interactions of polymyxin B, gramicidin S, and valinomycin with dimyristoyllecithin bilayers

The interactions of three polypeptide antibiotics (polymyxin B, gramicidin S, and valinomycin) with artificial lecithin membranes were studied by nuclear magnetic resonance (NMR). Combination of 31P and 2H NMR allowed observation of perturbations of the bilayer membrane structure induced by each of the antibiotics in the regions of the polar headgroups and acyl side chains of the phospholipids. The comparative study of the effects of these membrane-active antibiotics and the lipid bilayer structure demonstrated distinct types of antibiotic-membrane interactions in each case. Thus, the results showed the absence of interaction of polymyxin B with the dimyristoyllecithin membranes. In contrast, gramicidin S exhibited strong interaction with the lipid above the gel to liquid-crystalline phase transition temperature: disordering of the acyl side chains was evident. Increasing the concentration of gramicidin S led to disintegration of the bilayer membrane structure. At a molar ratio of 1:16 of gramicidin S to lecithin, the results are consistent with coexistence of gel and liquid-crystalline phases of the phospholipids near the phase transition temperature. Valinomycin decreased the phase transition temperature of the lipids and increased the order parameters of the lipid side chains. Such behavior is consistent with penetration of the valinomycin molecule into the interior of the lipid bilayers.     

Solvent history dependence of gramicidin-lipid interactions: a Raman and infrared spectroscopic study.

We have investigated the interactions between gramicidin and a model membrane composed of one phospholipid, dimyristoylphosphatidylcholine, as a function of the cosolubilization solvent and incubation time used in the sample preparation. Three organic solvents have been used; trifluoroethanol, a mixture of methanol/chloroform (1:1 v/v), and ethanol. Using Fourier transform infrared spectroscopy, we have demonstrated that the conformation adopted by gramicidin in the membrane is dependent upon the cosolubilization solvent used, and, only with trifluoroethanol, it is possible to incorporate gramicidin entirely as a beta 6.3-helix. Moreover, Raman spectroscopy results indicate that the orientation of the tryptophan side chains in gramicidin and their interaction with the hydrocarbon chains and the carbonyl groups of the lipids are also dependent on the cosolubilization solvent. On the other hand, the effect of the incorporation of gramicidin on the thermotropism of the lipid bilayer was found to be dependent upon the conformation of gramicidin in the lipid bilayers.     

The membrane as an environment of minimal interconversion. A circular dichroism study on the solvent dependence of the conformational behavior of gramicidin in diacylphosphatidylcholine model membranes

The conformation of gramicidin in diacylphosphatidylcholine model membranes was investigated as a function of the solvent in which peptide and lipid are initially codissolved. By use of circular dichroism it is demonstrated that, upon removal of the solvent and hydration of the mixed gramicidin/lipid film, it is the conformational behavior of the peptide in the organic solvent that determines its final conformation in dimyristoylphosphatidylcholine model membranes. As a consequence, parameters that influence the conformation of the peptide in the solvent also play an essential role, such as the gramicidin concentration and the rate of interconversion between different conformations. Of the various solvents investigated, only with trifluoroethanol is it possible directly to incorporate gramicidin entirely in the beta 6.3-helical (channel) configuration. It is also shown that the conformation of gramicidin in the membrane varies with the peptide/lipid ratio, most likely as a result of intermolecular gramicidin-gramicidin interactions at higher peptide/lipid ratios, and that heat incubation leads to a conformational change in the direction of the beta 6.3-helical conformation. Using lipids with an acyl chain length varying from 12 carbon atoms in dilauroylphosphatidylcholine to 22 carbon atoms in dierucoylphosphatidylcholine, it was possible to investigate the acyl chain length dependence of the gramicidin conformation in model membranes prepared from these lipids with the use of different solvent systems. It is demonstrated for each solvent system that the distribution between different conformations is relatively independent of the acyl chain length but that the rate at which the conformation converts toward the beta 6.3-helical configuration upon heating of the samples is affected by the length of the acyl chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-speed magic angle spinning solid-state 1H nuclear magnetic resonance study of the conformation of gramicidin A in lipid bilayers.

One- and two-dimensional solid-state 1H nuclear magnetic resonance spectra of gramicidin A incorporated in a dimyristoylphosphatidylcholine membrane have been obtained with use of high-speed magic angle spinning. By rotating the sample at 13 kHz, it is possible to observe signals in the 1H spectra between 6.0 and 9.0 ppm attributable to the aromatic protons of the tryptophan residues and the formyl group proton of gramicidin A. Two-dimensional solid-state COSY spectra provided information for the peak assignments. Moreover, changes in the 1H spectra have been observed as a function of the co-solubilization solvent initially used to prepare the samples and therefore as a function of the conformation adopted by gramicidin A. Three organic solvents have been used: trifluoroethanol, a mixture of methanol/chloroform (1:1 v/v), and ethanol. The conformational interconversion of gramicidin A from the double helix conformation to the channel structure for the sample prepared from ethanol was confirmed by following the time evolution of the proton spectra.     

External addition of gramicidin induces the hexagonal HII phase in dioleoylphosphatidylcholine model membranes

³¹P-NMR, small angle X-ray diffraction and freeze-fracture electron microscopy show that dioleoylphosphatidylcholine liposomes undergo a transition from the lamellar to the hexagonal H_II phase upon injection of an ethanolic solution of gramicidin in the aqueous medium, when the molar ratio of peptide to lipid is 1 to 20 or higher.     

X-ray scattering with momentum transfer in the plane of membrane. Application to gramicidin organization.

We demonstrate a technique for measuring x-ray (or neutron) scattering with the momentum transfer confined in the plane of membrane, for the purpose of studying lateral organization of proteins and peptides in membrane. Unlike freeze-fracture electron microscopy or atomic force microscopy which requires the membrane to be frozen or fixed, in-plane x-ray scattering can be performed with the membrane maintained in the liquid crystalline state. As an example, the controversial question of whether gramicidin forms aggregates in membrane was investigated. We used dilauroylphosphatidylcholine (DLPC) bilayers containing gramicidin in the molar ratio of 10:1. Very clear scattering curves reflecting gramicidin channel-channel correlation were obtained, even for the sample containing no heavy atoms. Thallium ions bound to gramicidin channels merely increase the magnitude of the scattering curve. Analysis of the data shows that the channels were randomly distributed in the membrane, similar to a computer simulation of freely moving disks in a plane. We suggest that oriented proteins may provide substantial x-ray contrast against the lipid background without requiring heavy-atom labeling. This should open up many possible new experiments.     

Conformational transitions of gramicidin A in phospholipid model membranes. A high-performance liquid chromatography assessment.

We have investigated the conformation of gramicidin A reconstituted in different phospholipid environments, small unilamellar vesicles, extensive bilayers, and micelles, by exploiting a recently proposed experimental approach based on high-performance liquid chromatography [Ba?ó et al. (1988) J. Chromatogr. 458, 105; Ba?ó et al. (1989) FEBS Lett. 250, 67]. The method allows the separation of conformational species of the peptide, namely, antiparallel double-stranded (APDS) dimers and beta 6.3-helical monomers, and quantitation of their proportions in the lipid environment. Various experimental parameters (e.g., nature of organic solvent, time of incubation in organic solvent, lipid-to-peptide mole ratio, time of sonication, and temperature) commonly involved in sample preparation protocols have been analyzed independently. The results show how the peptide conformation in model membranes is exquisitely dictated by the particular nature of the reconstitution protocol. In addition, we have elucidated the nature of the slow conformational transition of gramicidin toward the channel configuration that takes place upon incubation of the model membranes. This transition has been characterized as a temperature-dependent conversion from APDS dimeric to beta 6.3-helical monomeric forms. Analysis of kinetic data permits an accurate calculation of the rate constant for this process at different temperatures in phospholipid vesicles and micelles. Finally, an explanation is proposed for the laboratory-to-laboratory variation in the observed spectral patterns of inserted gramicidin.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.