INTRODUCTION

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Amphipathic peptides are promising candidates for the design of surfaces with well-defined properties. In particular, their enormous potential for forming self-organized monomolecular layers with a large variety of possible structures opens a challenging field in surface engineering. Among these structures are not only -helical and -strand structures, which are ubiquitous in biology, but also tailor-made structures like nanotubes (Ghadiri et al., 1994) and template-assembled peptides (TASPs) (Tuchscherer and Mutter, 1995). The assembly of the individual peptide molecules as a monolayer typically takes place at an appropriate interface (gas/solid, gas/liquid, or liquid/liquid) that serves as primary ordering template. The first successful attempts have been undertaken (Boncheva and Vogel, 1997; Kim et al., 1998), but for a rational design of peptide monolayers, a deeper understanding of their self-assembly principles is required. An attractive and simple possibility for manipulating the self-organization process is to control the surface pressure of a monolayer at the gas/liquid interface by Langmuir techniques. Indeed, the surface pressure behavior of various peptides is being intensively investigated, and many, partly contradictory attempts have been made to obtain structural information from these experiments. Surprisingly, relatively few studies deal with the direct determination of molecular conformation and orientation at the air/water interface. Besides x-ray and neutron reflection techniques (Berge et al., 1998; Lu and Thomas, 1998; Majewski et al., 1998; Naumann et al., 1996), one of the most promising approaches is infrared reflection absorption spectroscopy (Cornut et al., 1996; Flach et al., 1997; Gericke et al., 1997). However, in the latter case the problem of omnipresent and very strong water vapor bands must be overcome. These bands cover the spectral region of 1300-2000 cm¹, which also contains essential structural information originating from the amide I, amide II, carbonyl stretching, and methylene bending modes. An elegant way to overcome this problem is to selectively detect surface species by differential spectroscopy. This can be achieved by polarization modulation (PM) of the infrared light. This technique was originally developed for solid surfaces and has been successfully adopted to the spectroscopic characterization of molecules at the air/water interface within the last decade (Buffeteau et al., 1991; Blaudez et al., 1993, 1994, 1996; Cornut et al., 1996).

The present work concentrates on the orientation and conformation of gramicidin A at the air/water interface. Gramicidin A is a linear pentadecapeptide of alternating L- and D-amino acids (Sarges and Witkop, 1965) from Bacillus brevis that forms ion-selective membrane channels: formyll-X-Gly-L-Ala-D-Leu-L-Ala-D-Val-L-Val-D-Val-L-Trp-D-Leul-Trp-D-Leu-L-Trp-ethanolamine. Gramicidin adopts different conformations, depending on the environment and the pretreatment (Wallace, 1998). In lipid bilayers it is well established that it takes up a single-stranded ^6.3 structure (Ketchem et al., 1993, 1997; Wallace, 1992; Nabedryk et al., 1982; Urry, 1971). In contrast, in solution and in crystalline form, several different structures of intertwined helical dimers have been reported. Among them are antiparallel strands of ^5.6 helices (Langs, 1988) and ^7.2 helices (Wallace and Ravikumar, 1988), the latter as a cesium complex.

Here we present polarization-modulated infrared spectra of monolayers of pure gramicidin A as well as of mixtures with DMPC at the air/water interface. The experiments are complemented by simulations of polarization-modulated infrared spectra based on a well-established optical model (Yamamoto and Ishida, 1994; Mendelsohn et al., 1995). Recently, the strength of this approach has been evaluated thoroughly (Flach et al., 1997). To derive reliable information on the orientation of the peptide helix in the monolayer from the infrared (IR) spectra, it is crucial to know the direction of the transition dipole moment of the amide I band. More precisely, amide I bands typically comprise several modes that have to be taken into account, if they are of considerable intensity. In this work we address this problem by calculating PM-IRRAS spectra based on the normal mode calculations of Naik and Krimm (1986a), which provide a complete description of amide I bands for all possible conformations of gramicidin A.

	MATERIALS AND METHODS

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Gramicidin A was purchased from Fluka (Buchs, Switzerland). Dimyristoylphosphatidylcholine (DMPC) was obtained from Avanti Polar Lipids (Alabaster, AL). Water was purified with a MilliQ purification system and had a resistivity higher than 18 M cm. Deuterium oxide (D₂O) with 99.8% isotopic enrichment was supplied by Reactolab (Servion, Switzerland). High-performance liquid chromatography-grade methanol and chloroform were purchased from Fluka (Switzerland).

Gramicidin A, DMPC, and a 1:8 molar ratio mixture of the two were dissolved in methanol/CHCl₃ (1:1) to a final overall concentration of 1 mg/ml.

Pressure-area isotherms as well as PM-IRRAS measurements on H₂O as a subphase were carried out on a commercial film balance (Riegler and Kirstein, Berlin, Germany). Monolayers were formed by depositing a small amount of the solution on the surface of the water with a microliter syringe and allowing the solvent to evaporate. All measurements were performed at a temperature of 20°C. Experiments with D₂O were performed on a homemade miniaturized trough milled from Teflon with a subphase volume of only 10 ml. Because of the miniaturization, this trough had no facility for adjusting the surface area. Therefore, films were directly spread to the appropriate film pressure. For both troughs the film pressure was measured by the Wilhelmy plate method. The D₂O trough was enclosed in a plexiglass chamber (5 × 5 × 15 cm). The gas-tight chamber could be connected to two tubes sealed with BaF₂ windows, which led to the photoelastic modulator (PEM) and the ZnSe lens, respectively. Before spectra were acquired, this assembly was purged overnight with dry nitrogen. Then the trough was filled with D₂O with a syringe through a septum located on top of the plexiglass chamber. These measures were taken to keep the exchange of D₂O with H₂O as low as possible. Before film spreading, background spectra of the pure subphase were recorded.

PM-IRRAS spectra were recorded on a Vector 22 Spectrometer (Bruker, Karlsruhe, Germany) equipped with an external polarization modulation set-up (Fig. 1). The efficiency of the polarizer was specified as ranging from 98.2% (3000 cm¹) to 99.5% (1000 cm¹). The chosen angle of incidence was 75°, with an accuracy of ±1°. The photoelastic modulator (PEM-90; Hinds Instruments, Hillsboro, OR) modulated the polarization of the infrared light at a frequency of 74 kHz. Demodulation was performed with a lock-in amplifier (Princeton Applied Research, model 5209) and a low-pass filter (Stanford Research SR650). The optical velocity of the interferometer mirror was set at 0.47 cm/s. A total of 2000-5000 scans were recorded at 4 cm¹ resolution. Spectra were apodized with a triangular function and Fourier transformed with one level of zero filling. None of the spectra were smoothed.

View larger version (22K): [in this window] [in a new window]   FIGURE 1   Scheme of the PM-IRRAS set-up. The optical pathway encloses several plane mirrors, one off-axis parabolic mirror, a BaF2 wire grid polarizer, a photoelastic modulator, a ZnSe lens, and a MCT detector.

The principle of PM-IRRAS reflection absorption spectroscopy has already been described in detail elsewhere (Golden, 1985; Buffeteau et al., 1991; Hipps and Crosby, 1979). The measurable quantity, i.e., the polarization-modulated reflectivity S, is given as the ratio of the difference and the sum signal: where R_p and R_s are the reflectivities for polarization parallel and perpendicular to the plane of incidence and c is the electrical amplification ratio of the two signals. J₂ and J₀ are the second- and zero-order Bessel functions of the maximum dephasing angle ₀ that is introduced by the PEM. Spectral data are represented as where S and S₀ are the PM-IRRAS signals of the film-covered and film-free surface, respectively. This representation is independent of J₂ and C and reduces the large signal from the dielectric subphase. The resulting spectra can contain positive as well as negative bands, depending on the angle of incidence and the orientation of the transition moments. At the given angle of incidence of 75°, a positive reflection absorption band indicates a transition moment oriented preferentially in the plane of the surface, whereas a negative band indicates a transition moment oriented preferentially perpendicular to the surface. Intermediate transition moments give rise to a band with adjacent positive and negative components.

Simulation of spectra was performed using a self-developed computer program that computes the Fresnel reflection coefficients for parallel and perpendicular polarized light. There are several approaches described in the literature (Mendelsohn et al., 1995) that give virtually the same results. We implemented the one developed by Yamamoto and Ishida (1994). It is easily programmable and offers great flexibility because there are no restrictions concerning the number of layers. The mathematical formalism is based on the matrix method of Abelès (Born and Wolf, 1980), which describes stratified layers of homogeneous films. Appropriate modifications to account for absorbing (Dluhy, 1986; Hansen, 1968) and anisotropic (Yamamoto and Ishida, 1994) layers are included.

The final algorithm is valid for any system of stratified layers of absorbing, anisotropic, homogeneous material between a transparent semiinfinite incident medium and an absorbing, anisotropic, homogeneous, semiinfinite substrate.

Because the theory has been fully presented elsewhere (Yamamoto and Ishida, 1994), in the following description only the equations relevant to the computer program are summarized. Fig. 2 a gives a physical description on the basis of a three-phase system. The optical properties of the jth phase of the system are described by the anisotropic complex refractive indices in the c direction, where c represents x, y, or z coordinates: The characteristic matrices of the jth layer are defined as follows: where l represents perpendicular (s) or parallel (p) polarization and and with d_j represents the thickness and _jl the complex refractive angle of the jth layer. For an N-phase system (0  j  N  1) the overall matrix of the stratified layers results in The reflection coefficients may be derived from the elements m_ik of the matrix as The reflectivities of the film-free, R_l⁰, and film-covered, R_l, surfaces for parallel (l = p) and perpendicular (l = s) polarized light are calculated from the reflection coefficients using and To perform the simulation, values for _jc must be found. The directional extinction coefficients k_x max, k_y max, and k_z max of an anisotropic layer can be determined from the transition dipole strength k_max. Therefore the orientational distribution of the tilt angle, , between the main molecular axis and the surface normal (z axis) must be taken into account. Furthermore, assuming the transition dipolar moments to be equally distributed around the molecular axis at an orientational distribution (Fig. 2 b), the formalism of Fraser and MacRae (1973) for uniaxial symmetry in protein fibers applies: and with the orientational distribution order parameter where and reflect conformational fluctuations in the peptide and orientational fluctuations of the peptide in the monolayer, respectively. However, the orientational density functions that determine these distributions are unknown. In our calculations, we chose Dirac functions to describe the orientational density functions, resulting in  =  and  = .

View larger version (20K): [in this window] [in a new window]   FIGURE 2   (a) Schematic illustration of the optical model of a three-phase system. (b) Definition of the coordinate system. The rotational freedom of motion of the peptide around the z axis and of the transition moment around the molecular axis is shown. Symbols are described in the text.

The whole absorption band is expressed as an antisymmetrical linear combination of two Lorentzian functions (Ohta and Ishida, 1988): where fwhh is the full width at half-height, ₀ is the center wavenumber of the absorption band, is the actual wavenumber, and c represents x, y, z coordinates. The corresponding relations for the real refractive indices result from the Kramers-Kronig transformation of the last equation: where n_c is the constant refractive index in the near-infrared.

The angles  of the transition dipolar moments for amide I modes of the previously described structures of gramicidin A were derived from normal mode calculations (Naik and Krimm, 1986a). The results are shown in Table 1. The actual thickness of the gramicidin A layer was assumed to be between two limits defined by d  12.5 Å for the ^6.3 helix lying flat on the surface and d  31 Å for the ^5.6 helix standing upright. Hence, the thickness of the layer was far below the critical value where differences of the optical path due to reflection at the two interfaces come into play. Because, in this study, absorption is not considered in a quantitative manner, the absolute value of d as well as of the extinction coefficient k_max is not crucial. For the refractive index of the layers a value of n_c = 1.41 was chosen. Data for the complex refractive index of the H₂O or D₂O subphase were taken from the literature (Bertie et al., 1989) and extrapolated to the desired stepwidth.

View this table: [in this window] [in a new window]   TABLE 1   Relative intensities of amide I modes and angles of transition dipole moments, , for 6.3 and 5.6 conformations of gramicidin A as derived from normal mode calculations (Naik and Krimm, 1986a)

	RESULTS AND DISCUSSION

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS References

Monolayers of DMPC/gramicidin mixtures were prepared as described on the large as well as on the small Langmuir trough. DMPC and DMPC/gramicidin monolayers could easily be compressed or directly spread up to a lateral film pressure of 40 mN/m. No differences between the spectra were observed for the two spreading techniques. Thus in these cases the spreading procedure does not significantly influence structural features of the film that are detectable by IR spectroscopy.

Pure gramicidin layers

Pure gramicidin layers were typically examined at lower surface pressures, mainly for two reasons. First, gramicidin films at higher surface pressure are known to be extremely stiff, and the Wilhelmy plate tends to be pushed out of the subphase (Ducharme et al., 1996). Under these conditions the lateral film pressure cannot be measured precisely. Second, it is doubtful whether such rigid films could be produced by the direct spreading procedure due to the likely formation of collapse-like structures. However, on H₂O subphases we compressed gramicidin films to higher film pressures. The film was compressed to an area per molecule that was known to give the desired film pressure. The corresponding pressure-area isotherms made with a Langmuir-type measuring system were taken from the literature (Ducharme et al., 1996).

In Fig. 3, spectra of gramicidin on an H₂O subphase at three different film pressures, , are shown. For  = 14 mN/m and  = 25-30 mN/m, the amide I band is located at 1636-1638 cm¹, the amide II band at 1533-1535 cm¹. For  = 6 mN/m, the amide I band is shifted to slightly higher and the amide II band to slightly lower wavenumbers. Because of the very broad bands and the limited S/N ratio, a more precise determination of the wavenumbers is ambiguous.

View larger version (23K): [in this window] [in a new window]   FIGURE 3   PM-IRRAS spectra of the spectral region 1800-1400 cm1 of gramicidin A at the air/H2O interface at the indicated film pressures.

Based on these data, it is not possible to distinguish between the ^6.3 and ^5.6 helices from the location of the amide I band. Although a small difference in wavenumbers of the main peak is predicted from the normal mode calculations (Table 1) (Naik and Krimm, 1986a), experimental data gave a value of 1638 cm¹ for both structures (Naik and Krimm, 1986b; Nabedryk et al., 1982). The asymmetry of the measured amide I bands indicates the presence of a second component. We were not able to resolve this band precisely enough to decide whether its maximum is located at ~1650 cm¹ (^6.3 helix) or ~1670 cm¹ (^5.6 helix).

Experimental values for the amide II band have been found to be 1547 cm¹ for the ^6.3 helix (Nabedryk et al., 1982) and 1542 cm¹ for the ^5.6 helix (Naik and Krimm, 1986b), respectively. Neither of these values fits well with our findings, even though the agreement is better for the ^5.6 helix.

To elucidate the influence of the orientation of gramicidin within the layer on the amide I band, two sets of simulations were performed for the ^5.6 helix and the ^6.3 helix, respectively. The simulations are based on the values given in Table 1. The results are displayed for different tilt angles from 0° to 90° (Fig. 4).

View larger version (16K): [in this window] [in a new window]   FIGURE 4   Simulated PM-IRRAS spectra of the amide I band of gramicidin A at the air/H2O interface for tilt angles from  = 90° to  = 0° in steps of 15°. The refractive index of the film was n1 = 1.41. Other parameters were taken from Table 1. (a) Spectra for the 6.3 conformation. (b) Spectra for 5.6 conformation.

At a tilt angle of 90°, the spectra for both conformations are dominated by a single positive peak near 1640 cm¹. At lower tilt angles, the positive peak diminishes and a negative peak occurs. In the case of the ^6.3 helix, the positive peak vanishes completely and the negative peak appears at slightly higher wavenumbers. This result is in qualitative agreement with that obtained for the simulation of an -helix (Cornut et al., 1996), which is not surprising, because the angles of the main mode transition moments of 11° or 28°, respectively, are quite similar. In the case of the ^5.6 helix, the positive peak diminishes only moderately and the negative peak arises at much higher wavenumbers. From a comparison of the measured and simulated spectra, two conclusions can be drawn. First, with the present data it is not possible to clearly distinguish between ^5.6 and ^6.3 helices. Second, irrespective of the conformation, at  = 25-30 mN/m the tilt angle  of the gramicidin helix exceeds 60°.

Mixed DMPC/gramicidin layers

Fig. 5 displays the spectral region between 1800 and 1400 cm¹ for pure DMPC layers at 30 mN/m (Fig. 5 a), pure gramicidin layers at 14 mN/m (Fig. 5 b), and the DMPC/gramicidin mixture at 30 mN/m (Fig. 5 c), each on an H₂O subphase. The carbonyl stretching vibration at ~1733 cm¹, as well as the methylene scissoring vibration at ~1468 cm¹, of the lipid are clearly visible in spectra a and c. The spectrum of the pure gramicidin layer (Fig. 5 b) has already been described in the last section. It shows the amide I mode at ~1636 cm¹ and the amide II at ~1535 cm¹. The broad negative band in a and c at 1660 cm¹ stems from the (H₂O) bending mode of the liquid water subphase. Note that the spectrum of the pure gramicidin layer also contains a superimposition of the amide I band and the (H₂O) mode, although the latter is completely hidden by the amide band. The spectral region where the amide I band of the mixed layer (Fig. 5 c) is supposed to be located is completely dominated by the (H₂O) mode. Apparently, the intensity of the gramicidin amide I band is rather low. This is due to the following: 1) The gramicidin content in the mixed layer is only 11 mol%. 2) From Fig. 4, the intensity is also strongly dependent on the orientation of the helix.

View larger version (18K): [in this window] [in a new window]   FIGURE 5   PM-IRRAS spectra taken at the air/H2O interface of (a) a pure DMPC monolayer at 30 mN/m, (b) a pure gramicidin layer at 14 mN/m, (c) a DMPC/gramicidin A layer (8:1 molar ratio), and (d) weighted subtraction of spectrum a from spectrum c to eliminate (H2O).

At a first glance it may seem surprising that the spectral absorption of the putatively isotropic bulk water subphase is not eliminated by the polarization modulation. However, at and near the surface, the mean square electric field intensities in the x and y directions are reduced compared to the z direction because of interference between incident and reflected light (Dluhy, 1986), which implies a difference in absorption of parallel and perpendicular polarization. However, experimental (H₂O) bands show a considerably higher intensity than simulations that take account of this problem. Consequently, this spectral feature cannot be fully attributed to a difference in reflectivity between covered and uncovered water surfaces. The origin of this apparent discrepancy between experiment and simulation has been explained as being due to a thin layer of oriented water molecules beneath the surface layer (Blaudez et al., 1996). The properties of this water layer will strongly depend on the physical state and the chemical composition of the monolayer. The (H₂O) mode for the mixed DMPC/gramicidin layer might differ from that for the pure DMPC layer in both intensity and wavenumber. Consequently, it is not straightforward to extract the low-intensity amide I band signal in the mixed layer from the large water band. Nevertheless, the subtraction of the DMPC spectrum from the DMPC/gramicidin spectrum was carried out, and in such a way as to ensure complete elimination of the DMPC carbonyl stretching band. The resulting difference spectrum is shown in Fig. 5 d. A signalapparently consisting of a positive and a negative componentappears in the region where the amide I band of gramicidin should be found. However, artifacts may arise from the subtraction procedure, and the noise of the spectrum is substantially increased. For these reasons this signal could not be unambiguously assigned to the amide I band.

Therefore, to facilitate the interpretation of the spectra of mixed DMPC/gramicidin monolayers, the same measurements were performed on D₂O subphases. In these measurements the (H₂O) mode disappears and is replaced by the (D₂O) mode located near 1200 cm¹. In Fig. 6 the spectral region between 1800 and 1400 cm¹ is depicted for DMPC (Fig. 6 a), gramicidin (Fig. 6 b), and the DMPC/gramicidin mixture (Fig. 6 c) for D₂O subphases. As expected, the position of the CO stretching band in Fig. 6 a does not change significantly. The (H₂O) mode has vanished completely in spectra a and c. The very strong and broad band below 1500 cm¹ (Fig. 6, a-c) is mainly caused by the (HOD) mode. Hydrogen most probably originates as traces of H₂O that accumulate with time in the D₂O subphase.

View larger version (16K): [in this window] [in a new window]   FIGURE 6   PM-IRRAS spectra at the air/D2O interface of (a) a pure DMPC layer at 30 mN/m, (b) a pure gramicidin layer at 14 mN/m, (c) a DMPC/gramicidin A layer (8:1 molar ratio).

The amide I band of gramicidin (Fig. 6 b) is now located at ~1630 cm¹. It is shifted by 6 cm¹ to lower wavenumbers, as previously reported by other authors (Naik and Krimm, 1986b). The amide II band is shifted, because of the H-D exchange of the amide bond, to the region that is covered by the (HOD) mode.

Because the complex refractive index of D₂O differs from that of H₂O, the intensities of the bands for the two subphases are also different. In the wavenumber range under consideration, the bands for D₂O subphases are weaker than those for H₂O subphases. The CO stretching band of DMPC is of ~20-25% lower intensity (peak height) on D₂O than on H₂O, which is in good agreement with our simulations (20%). In contrast, the amide I band of the pure gramicidin layer is ~20% stronger than on an H₂O subphase, which can be attributed to the overlap of the amide I band on H₂O subphases with the negative (H₂O) mode. This compares well with simulations of the amide I band for D₂O subphases, which predict increases in intensity of 22% (^6.3 helix) and 25% (^5.6 helix). It implies that for the pure gramicidin layer the (H₂O) mode is as large as expected from the simulations.

In the spectrum of the mixed DMPC/gramicidin layer (Fig. 6 c), a band arises at ~1640 cm¹. This band can only be assigned to the amide I mode of gramicidin. Qualitatively, it coincides with the signal obtained from the difference spectrum for the H₂O subphase (Fig. 5 d). The signal-to-noise ratio is sufficient to allow a more quantitative analysis of the peak intensities. To do so, a series of spectra for DMPC/gramicidin monolayers were recorded at different film pressures in the range of 6-40 mN/m. The results are shown in Fig. 7 a. At low surface pressure the amide I modes of gramicidin form a positive absorption band. This band is rather broad, and its maximum occurs at ~1642 cm¹. With increasing surface pressure the band sharpens and gains intensity, and the maximum shifts to ~1625 cm¹. Subsequently, a negative band evolves at the expense of the positive band, resulting in an almost negative band at very high surface pressure.

View larger version (21K): [in this window] [in a new window]   FIGURE 7   (a) PM-IRRAS spectra of a DMPC/gramicidin A layer (8:1 molar ratio) at the indicated film pressures on a D2O subphase. (b) Simulated PM-IRRAS spectra of the amide I band of a DMPC/gramicidin A layer (8:1 molar ratio) at the indicated tilt angles, , on a D2O subphase.

As outlined in Materials and Methods, the positive absorption band at low surface pressure indicates a transition moment in the surface plane. Band shape and maximum are different from those obtained from pure gramicidin layers as well as from the mixed layers at higher surface pressures. This might be due to a different conformation of gramicidin at low surface pressures.

The formation of the negative band at higher surface pressure reveals that the orientation of the transition moment becomes increasingly perpendicular to the surface. To elucidate this point, we performed a series of simulations for the amide I band in the mixed layer for a variety of tilt angles of the peptide assuming ^6.3 conformation. The results of these simulations for the ^6.3 helix are presented in Fig. 7 b. A comparison of Fig. 7 a and Fig. 4 reveals that the ^5.6 conformation can be eliminated. It is clearly not consistent with the data. Neither the disappearance of the positive peak nor the appearance of the negative peak occurs at the correct wavenumber. The tilt angles in Fig. 7 b have been chosen to give the best fit to the measured spectra. The intensity of the simulated bands was scaled differently for each spectrum to reproduce the intensities of the measured bands reasonably well. A comparison of the measured and the simulated data clearly reveals the raising of the gramicidin helix upon compression.

A lipid monolayer with a surface pressure on the order of 30 mN/m represents the situation in a lipid bilayer (Jähnig, 1996). Therefore it is interesting to compare the tilt angle found for this particular film pressure with tilt angles found in lipid bilayers. With a refractive index of n = 1.41, we obtain a tilt angle of 31 ± 5°. The error was estimated on the basis of several measurements and takes account of the uncertainty in determining the ratio of positive and negative band components.

The tilt angle of 31 ± 5° is higher than what has been determined for gramicidin incorporated in DMPC vesicles ( = 15°) (Nabedryk et al., 1982). However, at that time no structural data were available for the ^6.3 helix, so the authors chose the transition moments from the then available structure of the ^4.4 helix. For this structure the transition moment was known to form an angle of 22° with the helix axis. Using the correct angle,  = 10.8°, we calculate from their data a tilt angle of 25°, which is in good agreement with our data.

The reliability of the tilt angle predictions presented here depends strongly on the tilt angle itself. For tilt angles  > 60°, the band consists of one single positive peak. In this situation the only measure of the tilt angle is the peak intensity. However, the intensity also depends on the absorption coefficient, the film thickness, and the surface coverage. Taking these additional factors into account introduces additional errors. In contrast, for 15° <  < 50°, taking the ratio of the intensities of the positive and negative components of the band, I₊ and I, eliminates the dependence on absolute intensities. In Fig. 8 the dependence of the ratio D_PM = |I₊/I| on  is shown. Similar graphs facilitating the determination of tilt angles from D_PM can be set up for different peptide conformations if  and n of the layer are known. However, one has to be aware of the fact that uncertainties in the estimation of refractive indices of the film and in the normal mode calculations might strongly affect the resulting tilt angle of the helix (Axelsen et al., 1995).

View larger version (17K): [in this window] [in a new window]   FIGURE 8   The ratio DPM of the positive and negative components of the PM-IRRAS spectra as a function of the tilt angle, , for the 6.3 conformation of gramicidin.

	CONCLUSIONS

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS References

PM-IRRAS in combination with simulations allowed us to study the conformation and orientation of gramicidin A at the air/water interface. If the angles of the contributing transition dipole moments with the molecular helix axis are known, the tilt angle of the helix at the water surface can be estimated from a single PM-IRRAS spectrum. In the present work the dipole moments could be derived from normal mode calculations. Within the applied pressure regime, the orientation of the pure peptide film seems to be only moderately dependent on the lateral pressure. In contrast, if the peptide is confined to a lipid monolayer, the orientation depends strongly on the lateral pressure, suggesting an alignment of the helix along the lipid chains. In this case, the lipid can be used as a matrix to orient the peptide in the desired manner. PM-IRRAS at the air/water interface has been shown to be a valuable tool for determining structural details of peptides at interfaces. This may accelerate the development of novel surface layers that are based on such amphiphilic peptides.