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Orientation and Lipid-Peptide Interactions of Gramicidin A in Lipid Membranes: Polarized Attenuated Total Reflection Infrared Spectroscopy and Spin-Label Electron Spin Resonance

Zoltán Kóta ^*, Tibor Páli ^* and Derek Marsh 

^* Institute of Biophysics, Biological Research Centre, Szeged, Hungary; and Max-Planck-Institut für biophysikalische Chemie, Abt. Spektroskopie, 37077 Göttingen, Germany

Correspondence: Address reprint requests to Dr. Tibor Páli, Institute of Biophysics, Biological Research Centre, PO Box 521, 6726 Szeged, Hungary. E-mail: tpali{at}nucleus.szbk.u-szeged.hu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS THEORETICAL BACKGROUND RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Gramicidin A was incorporated at a peptide/lipid ratio of 1:10 mol/mol in aligned bilayers of dimyristoyl phosphatidylcholine (DMPC), phosphatidylserine (DMPS), phosphatidylglycerol (DMPG), and phosphatidylethanolamine (DMPE), from trifluoroethanol. Orientations of the peptide and lipid chains were determined by polarized attenuated total reflection infrared spectroscopy. Lipid-peptide interactions with gramicidin A in DMPC bilayers were studied with different spin-labeled lipid species by using electron spin resonance spectroscopy. In DMPC membranes, the orientation of the lipid chains is comparable to that in the absence of peptide, in both gel and fluid phases. In gel-phase DMPC, the effective tilt of the peptide exceeds that of the lipid chains, but in the fluid phase both are similar. For gramicidin A in DMPS, DMPG, and DMPE, the degree of orientation of the peptide and lipid chains is less than in DMPC. In the fluid phase of DMPS, DMPG, and DMPE, gramicidin A is also less well oriented than are the lipid chains. In DMPE especially, gramicidin A is largely disordered. In DMPC membranes, three to four lipids per monomer experience direct motional restriction on interaction with gramicidin A. This is approximately half the number of lipids expected to contact the intramembranous perimeter of the gramicidin A monomer. A selectivity for certain negatively charged lipids is found in the interaction with gramicidin A in DMPC. These results are discussed in terms of the integration of gramicidin A channels in lipid bilayers, and of the interactions of lipids with integral membrane proteins.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS THEORETICAL BACKGROUND RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Gramicidin A is a 15-residue hydrophobic peptide (Fm-VGALAVVVWLWLWLW-Etn) that is composed of alternating D- and L-amino acids, which promote the formation of ß-helices. The peptide displays structural polymorphism, depending on the solvent, but a head-to-head dimer of ß^6.3 single helices is known to be the unit that forms ion-conducting channels in lipid-bilayer membranes. It is the latter that is the basis for the antibiotic activity of gramicidin A. A high-resolution structure of the ß^6.3-helix dimer has been determined in oriented lipid bilayers (Ketchem et al., 1993, 1997). This conformation is characterized by a ring of four tryptophan residues that are located at the membrane-water interface.

Depending on solvent conditions, gramicidin A may additionally form antiparallel ß^7.2 double helices (Wallace and Ravikumar, 1988). In principle, a variety of both single and double helices is possible structurally. Reconstitution in dimyristoyl phosphatidylcholine (DMPC) membranes from trifluoroethanol (TFE) produces the ß^6.3 single-helical form, but reconstitution from chloroform-methanol causes admixture of the nonconducting ß^7.2 double-helical form (Bouchard and Auger, 1993). Prolonged incubation tends to promote interconversion between these forms (Vogt et al., 1994).

The relatively small size and known structure of gramicidin A makes this a useful system for biophysical studies of membrane incorporation and lipid-protein interactions. Extensive studies have been made of the influence of gramicidin on lipid polymorphism (Killian, 1992), and of the ordering effects on the lipid chains (Rice and Oldfield, 1979; Killian, 1992). The effects on lipid dynamics have been investigated by spin-label electron spin resonance (ESR) spectroscopy (Patyal et al., 1997; Ge and Freed, 1999). Additionally, molecular dynamics (MD) simulations have been performed to characterize lipid interactions with the peptide, on a submolecular scale (Woolf and Roux, 1994, 1996).

Understanding the behavior of membranous antibiotics in bilayers of various lipids is key to the synthesis of new hydrophobic peptides with the desired membrane selectivity. Here, we study the integration of gramicidin A in bilayer membranes composed of different zwitterionic and anionic lipids, by orientation measurements with attenuated total reflection (ATR) infrared spectroscopy. There is considerable uncertainty in the literature as regards the orientations, _M, of the infrared transition moments of gramicidin, which are essential for determining the orientation of the peptide in the membrane (Nabedryk et al., 1982; Bouchard and Auger, 1993; Ulrich and Vogel, 1999). This problem is resolved here by spectral simulations, using the intensities and frequencies from normal mode calculations by Naik and Krimm (1986a). Further, we also investigate the stoichiometry and selectivity of lipid interactions with gramicidin A systematically by using different spin-labeled lipid species, in conjunction with ESR spectroscopy. Gramicidin A is an ideal model membrane protein with which to investigate selectivity of interactions with lipid headgroups that are not attributable to charged residues, because anchoring of the hydrophobic transmembrane peptide arises solely from interfacial tryptophan side chains (Hu and Cross, 1995). A particular goal of the ESR studies is to determine the possible role of interfacial tryptophan residues in lipid selectivity and in lipid-protein interactions in general. Samples are prepared from TFE to ensure formation of the channel structure, as characterized by infrared spectroscopy (Bouchard and Auger, 1993). It is found from spin-label spectroscopy that the lipid-peptide interactions in samples prepared from chloroform-methanol differ significantly from those prepared from TFE. Previous studies on gramicidin with spin-labeled lipids were devoted solely to the former method of preparation (Ge and Freed, 1999).

	MATERIAL AND METHODS

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS THEORETICAL BACKGROUND RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Materials
Gramicidin A was from Sigma Chemical Co. (St. Louis, MO). Phospholipids, DMPC, DMPS, DMPG, and DMPE were from Avanti Polar Lipids (Alabaster, AL). Spin-labeled stearic acid, 14-SASL, was synthesized according to Hubbell and McConnell (1971). Spin-labeled phosphatidylcholine, 14-PCSL, was synthesized by acylation of lysophosphatidylcholine with 14-SASL, as described in Marsh and Watts (1982). Other spin-labeled phospholipids, 14-PSSL, 14-PGSL, 14-PESL, and 14-PASL, were prepared from 14-PCSL by headgroup exchange mediated by phospholipase D (Marsh and Watts, 1982).

ATR spectroscopy
Polarized ATR infrared spectra were recorded on a Bruker (Karlsruhe, Germany) IFS-25 Fourier transform spectrometer at a resolution of 2 cm^-1. A horizontal ATR accessory from Specac (Orpington, U.K.), was used with a zinc selenide crystal (45° angle of incidence, six reflections). This was modified to seal the sample chamber hermetically from above, and to thermostat the cell housing with internally circulating water from a Haake (Karlsruhe, Germany) temperature-controlled bath. The surface of the ATR crystal available for coating by the sample was 8 x 45 mm. A germanium-mounted, wire-grid, linear polarizer (Specac) was used in the incident beam. Typically, 128–256 interferograms were co-added, and Fourier transformed after two levels of zero filling and apodization with a Blackman-Harris three-term function.

Samples containing 1.5–3 mg of lipid with 1:10 mol/mol gramicidin/lipid ratio were dried down on the ZnSe ATR crystal from a 10 mg/ml (DMPC), 3 mg/ml (DMPG and DMPE), or 1.5 mg/ml (DMPS) solution in TFE. Spectra of the dry sample were then recorded at the desired temperatures with radiation polarized parallel and perpendicular to the plane of incidence. The sample was then hydrated with 50 µl of D₂O (DMPC and DMPE), or of 10 mM Hepes, 2 mM EDTA, 1 M NaCl, pH 7.8, D₂O-buffer (DMPS and DMPG), by incubating above the chain-melting transition of the lipid bilayer, before again recording the polarized ATR spectra. Next, the sample was dried in a nitrogen gas stream for 12 h. Polarized spectra of the dry sample were then rerecorded. Finally, the sample was rehydrated with D₂O, or D₂O-buffer, and a second set of polarized spectra of the hydrated sample was recorded. For calculation of dichroic ratios, spectra from those dry and hydrated samples that exhibited the strongest dichroism were used. After baseline subtraction, bands in the amide I, II, and ester CO region (1520–1750 cm^-1), and in the CH stretching region (2830–2975 cm^-1), were fitted with Lorentzian components by using nonlinear least-squares minimization. The amide A region (3110–3640 cm^-1) was fitted with matching Gaussian bandshapes. Second-derivative spectra were used to provide initial estimates of the component band positions. Dichroic ratios were calculated by using the integrated intensities of the corresponding bands.

Molecular orientations are calculated from the measured dichroic ratios, R, according to (Marsh, 1999a):

(1)

where is the orientation of the molecular axis relative to the normal to the orienting substrate (i.e., the ATR plate), and is the orientation of the transition moment relative to the molecular axis. Angular brackets in Eq. 1 indicate summation over the (normalized) orientational distribution. The strengths of the electric field components, with a zinc selenide ATR crystal, are: in the thick film approximation, which is appropriate to the amount of material used (Marsh, 1999a). Order parameters, are also used to define the molecular orientations. These order parameters are a composite of the molecular orientation in the membrane and the degree of alignment of the membrane relative to the surface of the ATR crystal. Effective tilt angles, _eff, are obtained from by assuming a singular value of . Values of _eff obtained for the lipid chains in the gel phase are used to assess the degree of macroscopic alignment of the membranes, as described in the Results section.

The orientation of the transition moment is taken as _C = 90° for the methylene and carbonyl stretch bands of the lipid. The transition moment orientations for the amide I band of gramicidin A are considered in the following main section, based on the normal mode calculations of Naik and Krimm (1986a). A value of _I = 32°, corresponding to the maxima in the low frequency region of the amide I band, is chosen from bandshape simulations. This approach is necessary because of the strong overlap of the modes that is predicted for the ß^6.3-helix (see Theoretical Background).

ESR spectroscopy
ESR spectra were recorded on a Bruker (Karlsruhe, Germany) EMX 9-GHz spectrometer with rectangular cavity and nitrogen gas-flow temperature regulation. Samples were accommodated in 1-mm diameter glass capillaries, which were placed in a standard quartz ESR tube that contained light silicone oil for thermal stability. A solution of gramicidin A plus phospholipid and 1 mol% spin label (2 mg of lipid at 7 mg/ml) was dried down under nitrogen and then incubated under vacuum overnight. The dry sample was hydrated with excess buffer (70 µl of 10 mM Hepes, 2 mM EDTA, pH 7.8) by vortex mixing above the chain-melting temperature of the lipid. This was then pelleted in the glass capillary by using a benchtop centrifuge. Excess supernatant was removed and the capillary was sealed. For samples containing spin-labeled stearic acid (14-SASL), the buffer was either 10 mM Na acetate, 2 mM EDTA pH 4.5, or 10 mM Tris, 2 mM EDTA pH 9.0.

Analysis of the two-component ESR spectra was performed by spectral addition, using least-squares optimization. The two quasi-single component spectra were taken from samples of high lipid/peptide ratio, and low lipid/peptide ratio, respectively. Spectra of the latter were recorded at low temperature, below that of chain melting.

	THEORETICAL BACKGROUND

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS THEORETICAL BACKGROUND RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Orientation of the amide I transition moments of gramicidin A
For an isolated mode, the ratio of the intensities with radiation polarized parallel (I_||) and perpendicular (I) to the helix axis, can be calculated from the vector components of the transition moment (see, e.g., Marsh et al. (2000)):

(2)

where _M is the orientation of the transition moment, M, relative to the helix axis. Strict axial symmetry is assumed, which requires inclusion of the factor two in Eq. 2. Because the normal modes are independent, but their transition moments all transform in exactly the same way on rotation (see Eqs. 5 and 6, given in the next subsection), Eq. 2 applies equally to overlapping band components when summation is made over their contributions to the total intensities I_|| and I.

Normal mode calculations by Naik and Krimm (1986a) indicate that the most intense amide I band component of gramicidin A has A-species symmetry and is predicted to have the lowest frequency and preferentially parallel polarization. This is designated by A() in Table 1. However, other modes with different polarizations also contribute appreciable intensity in this region of the spectrum. The envelope of the amide I band with radiation polarized parallel or perpendicular to the helix axis that is predicted by the normal mode calculations of Naik and Krimm (1986a) is given in Fig. 1 for the ß^6.3 and ß^4.4 single-helix, and ß^7.2 and ß^5.6 antiparallel double-helix conformations. This figure was generated from the frequencies, intensities, and polarizations of the normal modes by assigning a Lorentzian lineshape with full-width at half-height (FWHH) of 15 cm^-1 to each mode. This value of the FWHH corresponds to that found experimentally for the intense amide I component of gramicidin A in DMPC (see later). It should be noted that the vibrational frequencies correspond to calculations for fully proteated amides in gramicidin A. For the deuterated species, these frequencies are 6 cm^-1 lower (Naik and Krimm, 1986b).

View this table: [in this window] [in a new window]   TABLE 1  Parallel and perpendicular polarized relative intensities (I|| and I)

View larger version (23K): [in this window] [in a new window]   FIGURE 1  Amide I bandshapes of proteated gramicidin A, for radiation polarized parallel (solid lines) and perpendicular (dashed lines) to the helix axis, simulated by a sum of Lorentzians of width 15 cm-1 centered at the component frequencies from normal mode calculations by Naik and Krimm (1986a). (A) ß6.3 single helix and ß7.2 double-stranded helix, as indicated. (B) ß4.4 single helix and b5.6 double-stranded helix, as indicated.

It is seen from Table 1 that, with the exception of the ß^5.6-helix, the effective orientations of the transition moment ( 31–33°) are rather similar when deduced from the local maxima in the low frequency region of the spectrum. These differ considerably, however, from values previously assumed for gramicidin A. Nabedryk et al. (1982) assumed a value of _I = 22° based on the geometry of a ß^4.4-helix and this value was adopted also by Bouchard and Auger (1993). This estimate lies close to that given for the most intense mode of the ß^4.4-helix in Table 1. On the other hand, Ulrich and Vogel (1999) have advocated the use of the value _I = 10.8° that corresponds to the most intense mode of the ß^6.3-helix (see Table 1). This value is inappropriate for the experimental spectra, however, because of the strong overlap with nearby unresolved modes, as represented by the band envelopes shown in Fig. 1.

Also given in Table 1 are the values of the intensities, and corresponding effective values of _I, that are obtained from summation over the entire amide I band. These are designated by A(tot). They may be appropriate, if the absorbance is obtained by spectral integration, without band fitting.

Simulation of dichroic spectra from gramicidin A
For a given degree of orientational order of gramicidin A, prediction of the bandshapes in the amide I region of the polarized ATR spectra, is possible also by using frequencies and intensities from the normal mode calculations of Naik and Krimm (1986a). The absorbances for parallel (A_||) and perpendicular (A) polarized radiation in the ATR experiment are given by (see, e.g., Marsh (1997a)):

(3)

(4)

where M_i = (M_x,i, M_y,i, M_z,i) is the transition moment of the ith amide I mode and k is a constant. Angular brackets indicate summation over all molecular orientations. The z-axis is normal to the plane of the ATR crystal and the x- and y-axes lie within and orthogonal to the incident beam, respectively. With the expressions for the components of M_i given in Marsh (1997a), and assuming axial symmetry, the absorbance for the two polarizations is given by:

(5)

(6)

where and are the intensities of the amide I modes given by Naik and Krimm (1986a). The total absorbance in the amide I region is then given by summation over all modes, i. To do this, a Lorentzian bandshape of given width is associated with each mode.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS THEORETICAL BACKGROUND RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Polarized ATR spectra
FTIR spectra were recorded with radiation polarized parallel or perpendicular to the plane of incidence. Fig. 2 shows typical polarized spectra for gramicidin A in aligned DMPC membranes. Dichroism is observed both in the lipid chain methylene bands and in the amide bands of gramicidin, as seen from the differences between the solid and dashed spectra for the indicated bands. Little intensity is observed in the amide II region of the spectrum, demonstrating that all amides are accessible to H-D exchange with D₂O. Table 2 lists the dichroic ratios of the amide I, amide A, lipid carbonyl, and methylene stretch bands, for both dry and hydrated samples, at temperatures in the gel and fluid phases of DMPC bilayers. These ratios, R = A_||(tot)/A(tot), were determined from the integrated intensities, A(tot), of the different bands. Rehydration, after drying the hydrated samples, improved the degree of alignment in the hydrated state. Therefore, the final two stages of the repeated drying and hydration cycles in sample preparation are reported in Table 2 (see also Materials and Methods). It is seen that, in most cases, the dichroic ratios for dry and hydrated samples are rather similar, which is only possible for the thick film approximation. Table 2 includes values of the order parameters, P₂(cos ), that are calculated from Eq. 1 by using the electric field intensities given in the Materials and Methods section. In the dry state, all order parameters remain essentially constant with increasing temperature, whereas the lipid order parameters of the hydrated membranes decrease at the chain-melting transition, from the gel phase at 10°C to the fluid phase at 36°C. The highest order parameters in Table 2 are obtained for the lipid chains in the hydrated gel phase, at 10°C. Taking account of the fact that the lipid chains are tilted in the gel phase of DMPC (Marsh, 1990), these lipid chain order parameters show that the membrane normal is oriented almost perpendicular to the orienting surface of the ATR crystal (see Discussion).

View larger version (22K): [in this window] [in a new window]   FIGURE 2  Polarized ATR spectra from aligned dimyristoyl phosphatidylcholine membranes containing gramicidin A at a lipid/peptide ratio of 10:1 mol/mol. Solid and dashed lines are for radiation polarized parallel and perpendicular to the plane of incidence, respectively. (Top) Dry membranes; (bottom) membranes hydrated with excess D2O. Temperature, 10°C.

View larger version (32K): [in this window] [in a new window]   FIGURE 3  Amide I band in polarized ATR spectra from hydrated aligned phospholipid membranes containing gramicidin A at a lipid/peptide ratio of 10:1 mol/mol. Solid and dashed lines are for radiation polarized parallel and perpendicular to the plane of incidence, respectively. (A) In phosphatidylcholine (DMPC) membranes; (B) in phosphatidylserine (DMPS) membranes; (C) in phosphatidylglycerol (DMPG) membranes; and (D) in phosphatidylethanolamine (DMPE) membranes. Membranes are hydrated in D2O (DMPC and DMPE), or in D2O buffer: 10 mM Hepes, 1 M NaCl, pH 7.8 (DMPS and DMPG); temperature 10°C. Dotted lines are the components obtained from band fitting, except those overlaying the solid and dashed lines, which are the sum of the fitted components.

Simulation of dichroic spectra
The amide I band region in the polarized ATR spectra of gramicidin A in DMPC, was simulated by using the experimental order parameter, P₂(cos ), and the normal mode results of Naik and Krimm (1986a), as described in the section on Theoretical Background. This gives a check on consistency, because the value of P₂(cos ) was determined simply from the dichroic ratio and an effective value of the transition moment orientation, and not from the overall bandshape. Additionally, the bandshape simulations afford further possibility to distinguish between the channel-forming and double-helix conformations of gramicidin.

The amide I band envelopes predicted according to Eqs. 5 and 6 from the normal mode analysis for the gramicidin A ß^6.3-single helix and the ß^7.2-double-stranded helix are given in Fig. 4, A and B, respectively. For this, a Lorentzian lineshape with full width at half-height (FWHH) of 15 cm^-1 is associated with each mode. The latter corresponds to the FWHH of the intense component at 1630 cm^-1 in the experimental spectrum of gramicidin A in DMPC (see above). Amide I bandshapes are given for radiation polarized parallel (solid lines) and perpendicular (dashed lines) to the plane of incidence, by using the thick film approximation. Corresponding calculations with the thin film approximation are given by dotted lines.

View larger version (20K): [in this window] [in a new window]   FIGURE 4  ATR amide I bandshapes for proteated gramicidin A predicted from Eqs. 5 and 6 and the normal mode analysis of Naik and Krimm (1986a), (A) for the ß6.3 single-helix conformation, and (B) for the ß7.2 double-stranded helix conformation. The thick film approximation is used with a helix tilt of eff = 39° (cos2 = 0.60) to obtain the ATR absorbance with parallel (solid line) and perpendicular (dashed line) polarized radiation. (Dotted lines are corresponding calculations with the thin film approximation, eff = 30°; cos2 = 0.75). Each component mode has a Lorentzian lineshape with FWHH = 15 cm-1.

Quantitation by peak fitting to the parallel and perpendicular polarized spectra given in Fig. 3 (see Marsh (1999b)) reveals that the high-frequency shoulder constitutes 30% of the total intensity of the amide I band. This would correspond to 4 residues per gramicidin monomer (including the blocked N- and C-termini). This is not unreasonable for the effective number of end residues whose H-bonding does not conform strictly to the pattern for an infinite ß-helix. Three residues at the membrane surface are hydrogen bonded to water and three residues at the dimer interface are involved in H-bonding to the partnering monomer (Buchet et al., 1985). For the former, the amide I band components are expected in the region of 1640–1650 cm^-1. The residues at the dimer interface are arranged as antiparallel ß-strands. For these a weak band is expected at 1670–1680 cm^-1 and a strong band at 1630–1640 cm^-1, much as for the antiparallel double helices (see Theoretical Background). This is in accord with the band fitting for the amide I region of gramicidin A in DMPC that was given above.

Lipid spin-label ESR
The ATR results of the previous section show that best orientation of gramicidin A was obtained in a DMPC host membrane. Therefore, this lipid was chosen to study the lipid-peptide interactions further by using spin-labeled lipids at probe concentrations. Fig. 5 gives the ESR spectra of phosphatidylcholine spin-labeled at the 14-position of the sn-2 chain (14-PCSL) in DMPC membrane dispersions that contain gramicidin A at different lipid/peptide molar ratios. The sample temperature is 30°C, at which DMPC membranes are in the fluid phase. For gramicidin/DMPC ratios >1:20 mol/mol, a second component with increased hyperfine splitting appears in the outer wings of the ESR spectra. This component corresponds to a lipid population with chain mobility that is more restricted than that in fluid lipid bilayers. Its intensity increases progressively, relative to that of the fluid-bilayer lipid population, as the content of gramicidin A in the membrane increases. Quantitation of the fraction, f, of motionally restricted lipids was performed as described in the Materials and Methods section. Assuming that the spin-label distribution directly reflects that of unlabeled PC (i.e., K_r 1), the motionally restricted lipid population corresponds to N_b = 3.6 ± 0.3 lipids/gramicidin, consistently over the lipid/peptide range: 5:1–15:1 mol/mol.

View larger version (17K): [in this window] [in a new window]   FIGURE 5  ESR spectra of 14-PCSL phosphatidylcholine spin label in membranes of DMPC containing gramicidin A at the lipid/peptide molar ratios indicated on the figure. Buffer, 10 mM Hepes, 2 mM EDTA, pH 7.8; T = 30°C.

(7)

where f^PC is the value of f for 14-PCSL (see, e.g., Marsh and Horváth (1998)). The selectivity ranking is: SA(pH 9)>PAPS>PC>PGPE>SA(pH 4.5), for samples prepared from either solvent.

View larger version (24K): [in this window] [in a new window]   FIGURE 6  ESR spectra of spin-labeled phospholipids: 14-PCSL, 14-PSSL, 14-PESL, 14-PASL, and 14-PGSL, in DMPC/gramicidin A (10:1 mol/mol) membranes hydrated with 10 mM Hepes, 2 mM EDTA, pH 7.8. The bottom two spectra are from spin-labeled stearic acid, 14-SASL, at pH 4.5 (10 mM Na acetate, 2 mM EDTA) and pH 9.0 (10 mM Tris, 2 mM EDTA), as indicated. (Left) Samples prepared from TFE; (right) samples prepared from chloroform-methanol (2:1 v/v). T = 30°C.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS THEORETICAL BACKGROUND RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

Dichroic ratios for gramicidin A in DMPC in the dry state have been measured by polarized transmission spectroscopy (Nabedryk et al., 1982). The samples were prepared from chloroform. Values of _M were predicted from molecular coordinates for a ß^4.4-helix and measurements on an -helical model compound. The maximum inclination of the amide I transition moment to the helix axis that is calculated from the experimental data of Nabedryk et al. (1982) is _I = 27 ± 1°, assuming that the sample is perfectly oriented (i.e., _eff = 0°). Whereas this value is within the limits set for the most intense mode of a ß^6.3-helix, it is less than the effective values of _I (32°) derived from the band maxima in the low-frequency region (see Table 1). One conclusion that can be drawn from this is that the samples of Nabedryk et al. (1982) are highly ordered. The other conclusion is that the effective orientations of the transition moment derived from the band maxima predicted by the normal mode analysis of Naik and Krimm (1986a) may be upper estimates.

Dichroic ratios have also been measured previously for gramicidin A in fluid hydrated DMPC bilayers by polarized ATR (Bouchard and Auger, 1993). Using the orientations of the transition moment given in the section on Theoretical Background (i.e., _I = 32°), rather than the older ones referred to above, yields values of P₂(cos ) = 0.39 ± 0.06 and _eff = 40 2° for a sample prepared from TFE and incubated for 4 h. These values are in good agreement with those given in Table 2, which were obtained also from samples prepared from TFE but with a different ATR crystal.

The order parameter for the lipid chains in the gel phase at 10°C that is given in Table 2 corresponds to an effective tilt of _eff = 33° (CH₂ symmetric stretch), when a unique orientation is assumed. For DMPC alone, a similar value of _eff = 29° was obtained (data not shown). This is comparable to the chain tilt of DMPC in the L_ß' phase obtained from x-ray diffraction studies (30°, see, e.g., Marsh, 1990). From this, one can conclude that the DMPC membranes are reasonably well aligned. Nevertheless, the effective tilt of the gramicidin A molecule at 10°C somewhat exceeds this value. Possibly the peptide channel cannot incorporate perfectly in a single domain of the tilted lipid gel phase and may be associated with regions of azimuthal disorder in the chain-tilt direction. Note, however, that gramicidin A is not excluded from the lipid gel phase. Our preliminary results from differential scanning calorimetry show that the DMPC pretransition shifts progressively to lower temperature with increasing content of gramicidin A.

In the fluid phase, the effective tilt of the lipid chains increases (i.e., the order parameter decreases), relative to the gel phase. This chain disorder results from trans-gauche isomerism about the C-C bonds in the lipid chains. Under these conditions, the order parameter of gramicidin A, however, does not decrease. Instead, the order improves in the fluid phase. The hydrophobic span and effective tilt of gramicidin A match more closely those of the disordered lipid chains than for the extended chains in the gel phase (see Table 2). One, therefore, can conclude that the peptide incorporates well in the fluid L phase of hydrated DMPC.

Orientation of gramicidin A in different lipids
Fig. 7 gives the effective tilt angles, _eff, of gramicidin A (amide I) and the lipid chains (CH₂ symmetric stretch) in the different phospholipid hosts tested. Data are given for both gel and fluid phases of the respective lipid bilayers. These values increase (i.e., the degree of orientation decreases) progressively from DMPS, via DMPG, to DMPE. With the exception of the fluid phase of DMPS, the tilt-values are also greater than for DMPC. Concomitantly, the degree of disorder of gramicidin A exceeds that of the lipid chains, in the fluid phase. This is unlike the situation for the DMPC host. The effect is particularly dramatic in the case of DMPE, where gramicidin A is almost completely disordered under all conditions. The dichroic ratios are then close to the isotropic value, R_ISO = 2, and _eff lies close to the magic angle. This can be interpreted as indicating that gramicidin A incorporates less well into membranes of the negatively charged lipids, DMPS and DMPG. Most likely it incorporates hardly at all into the weakly hydrated DMPE membranes, despite being entirely devoid of charged or polar residues. Internal hydrogen bonding within the DMPE surface layer (Elder et al., 1977), and its low hydration, presumably precludes hydrogen bonding of the ester carbonyls with the interfacial tryptophan residues, which is an important feature of membrane integration for gramicidin A (Woolf and Roux, 1994; Hu and Cross, 1995).

View larger version (39K): [in this window] [in a new window]   FIGURE 7  Effective orientation, eff, deduced from ATR order parameters of the lipid chains (CH2 symmetric stretch; left two bars for each), and of gramicidin (amide I; right two bars for each), in fully hydrated membranes of DMPC, DMPS, DMPG, and DMPE with a lipid/peptide ratio of 10:1 mol/mol. Data are given for temperatures in the gel (left bar in each pair) and fluid phases (right bar in each pair) of each lipid: DMPC, 10°C and 40°C; DMPS, 24°C and 50°C; DMPG, 10°C and 40°C; and DMPE, 37°C and 63°C.

From Fig. 6, it is evident that the lipid stoichiometry in samples prepared from chloroform-methanol is greater than that in samples prepared from TFE. In the former case, the motionally restricted population, f, of 14-PCSL corresponds to six lipids per gramicidin A monomer (see Table 6). The difference is attributable to the fact that samples prepared from chloroform-methanol are composed, at least in part, of the ß^7.2 double helix (Bouchard and Auger, 1993). This configuration is fully transmembrane and therefore might be more effective in restricting the motion of spin labels close to the terminal methyl group of the lipid chains than is the head-to-head dimer. Additionally, both any putative aggregation pattern and the exact disposition of the interfacial tryptophans may differ for this conformer. Indeed, the solvent history generally could affect the aggregation state of gramicidin A in the membrane.

Although gramicidin A is a hydrophobic peptide, devoid of any strongly polar residues, the results given in Table 6 demonstrate that it does express a selectivity of interaction between different lipid species. Previous ESR experiments have demonstrated that the selectivity for negatively charged lipids, which is displayed by a range of integral membrane proteins, is not solely electrostatic in origin (Marsh and Horváth, 1998). The lipid headgroup selectivity that is exhibited by gramicidin A is of the non-Coulombic type. Almost certainly it involves the strongly dipolar properties of the interfacial tryptophans that are present in both the ß^6.3 and ß^7.2 structures.

It is of interest to compare the lipid selectivities at the peptide-lipid interface that are determined by ESR in DMPC with the results on the integration of gramicidin A in different host lipid species that was explored by ATR. Incorporation of gramicidin into lipid membranes depends not only on the specificity of lipid interactions locally at the lipid-peptide interface, as measured by different spin-labeled lipid species at probe concentrations. It depends also on the intrinsic properties of the bilayers of the host lipid, i.e., on the lipid-lipid interactions in the membrane environment into which the peptide must incorporate. For this reason, the two sets of measurements are not necessarily equivalent. Evidently, it is lipid-lipid interactions, and not direct lipid-peptide interactions, that dominate the energetics of peptide incorporation into certain lipids.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS THEORETICAL BACKGROUND RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
In addition to the role of the lipid chains, which has been investigated previously (Killian, 1992), the lipid polar groups and surface hydration also play a significant part in the successful integration of gramicidin into the lipid bilayer. Interfacial tryptophan residues in gramicidin A are important for both the stoichiometry and selectivity of lipid interactions with this peptide. This could have several implications for the interactions of lipids with integral transmembrane proteins. Tryptophan residues might quite generally nucleate the formation of crevices that could accommodate lipids at the surface of integral membrane proteins. In addition, they could be a major source of lipid selectivity that is not based directly on the net charge of the lipid headgroups—selectivity of non-Coulombic origin is a feature that is quite widespread amongst integral proteins (Marsh and Horváth, 1998).

	ACKNOWLEDGEMENTS

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS THEORETICAL BACKGROUND RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
We thank Frau B. Angerstein for synthesis of spin-labeled lipids.

This work was supported in part by the Volkswagen-Stiftung, the Hungarian National Science Fund (T029458 and T043425), and a German-Hungarian travel grant (DAAD-MOB 18/2001). D.M. and T.P. are members of the European Union COST D22 Action.

	FOOTNOTES

 
Abbreviations used: ATR, attenuated total reflection; DMPC, DMPE, DMPG, and DMPS, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, -phosphoethanolamine, -phosphoglycerol, and -phosphoserine; EDTA, ethylenediaminetetraacetic acid; ESR, electron spin resonance; Hepes, N-(2-hydroxyethyl)piperazine-N'-2-ethanesulphonic acid; MD, molecular dynamics; 14-PASL, 14-PCSL, 14-PESL, 14-PGSL, and 14-PSSL, 1-acyl-2-(14-(4,4-dimethyl-oxazolidine-N-oxy))stearoyl-sn-glycero-3-phosphoric acid, -phosphocholine, -phosphoethanolamine, -phosphoglycerol, and -phosphoserine; 14-SASL, 14-(4,4-dimethyl-oxazolidine-N-oxy)stearic acid; TFE, trifluoroethanol; Tris, tris-hydroxymethylaminoethane.

Submitted on November 22, 2002; accepted for publication October 21, 2003.

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