INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS DATA ANALYSIS RESULTS DISCUSSION CONCLUSION REFERENCES

The phase transition of phospholipid monolayers at the air/water interface between a fluid-like liquid expanded (LE) and a more solid-like liquid condensed (LC) phase is a well-known phenomenon and has been studied in great detail over the past two decades (Albrecht et al., 1978; Möhwald, 1995). The transition from the LE to the LC phase is connected with the formation of micron size, crystal-like domains at lateral pressures above the transition point, which can be readily observed by fluorescence or Brewster angle microscopic methods (Möhwald, 1995). For monolayers consisting of more than one lipid species, the domain formation may lead to a partial demixing of the lipids, i.e., an enrichment of one species within the domain at the expense of the other species. If one of these species carries an excess electric charge while the other is zwitterionic or neutral, the demixing is equivalent to the creation of a heterogeneous distribution of surface charge density over the monolayer. In contrast, under LE phase conditions, this charge distribution can be expected to be homogenous owing to the lateral diffusion of the lipids (Peters and Beck, 1983).

This change of surface charge distribution between LE and LC phase may lead to differences in the partition of water-soluble proteins undergoing a Coulomb attraction by the monolayer between the surface and the subphase. Thus, it seems feasible that, under certain conditions, electrostatic protein binding and unbinding to or from the monolayer can be controlled by its phase state. For phospholipid bilayers on a solid support, we demonstrated recently that this concept of the modulation of protein binding by the lipid phase state is indeed functional (Käsbauer and Bayerl, 1999) and can be used for the advanced separation (phase transition chromatography) of proteins (Loidl-Stahlhofen et al., 1996).

The aim of this work is to provide evidence that a similar mechanism exists in lipid monolayers at the air/water interface and with proteins dissolved in the subphase. To detect differences in the amount of proteins attached to the monolayer as a function of its phase state, we used two surface-sensitive methods that are both well established for the study of monolayer systems: neutron reflection (NR) and infrared reflection-absorption spectroscopy (IRRAS). The introduction of a trough shuttle technique for IRRAS, as previously suggested by Flach et al. (1994), allowed, in our setup, for an optimum compensation of rotational water-absorbance bands. This enabled reproducible measurements of protein adsorption to monolayers over a long time range (up to 10 h). The combined application of these methods allows detailed information about the structure and density of the bound protein layer to be obtained and thus gives information on the amount of protein bound to the monolayer. The water-soluble protein selected for this study was cytochrome c because it has been used in the previous bilayer studies (Käsbauer and Bayerl, 1999) and a detailed knowledge is available on its structure and electrostatic nature of its interaction with lipid surfaces.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS DATA ANALYSIS RESULTS DISCUSSION CONCLUSION REFERENCES

Materials

All lipids were purchased from Avanti Polor Lipids (Alabaster, AL) and were dissolved in a 9:1 chloroform/methanol solution at a concentration of 1 nmol/µl. From these solutions, the binary mixtures listed in Table 1 were prepared volumetrically.

All experiments involving cytochrome c were carried out using a subphase of D₂O (for NR, 99.9% purity from Fluorchem Ltd., Old Glossop, UK; for IRRAS, 97.5% purity from Deuchem GmbH, Leipzig, Germany), which was buffered at pH 7.0 ± 0.1 with 20 mM HEPES and 0.25 mM EDTA. Experiments on pure lipid monolayers were performed using D₂O and H₂O (Millipore purified water) buffered subphases as described above.

Cytochrome c was obtained from Fluka (Deisenhofen, Germany) and was dissolved in an aliquot of the buffer at concentrations of up to 2 mg/ml. The dissolution was done at least 2 h in advance of the NR or IRRAS experiments to allow labile protons to exchange with deuterons from the buffer.

Lipid monolayers were spread from the organic solution using a microsyringe and, afterwards, compressed to the desired surface pressure. For both the NR and the IRRAS experiments, Langmuir troughs were used, which allow the pressure-tight sealing of an inner "protein compartment" after compression by closing a 2-mm wide channel link with a teflon plate (Naumann et al., 1996). This procedure avoids film leakage and allows a better control of the protein concentration in the subphase.

The maximum subphase surface area of the Langmuir troughs was 289 cm² and 223 cm² for the NR and IRRAS experiments, respectively. The inner protein compartments had areas and volumes of 64.5 cm² and 38.5 ± 2.5 ml for NR and of 49.7 cm² and 27.5 ± 2.5 ml for IRRAS. The subphase was kept at 20.0 ± 0.2°C for all experiments.

Methods

NR was measured at the CRISP spectrometer of the ISIS spallation source (Rutherford-Appleton Laboratory, Chilton, U.K.) according to procedures previously described in detail (Maierhofer and Bayerl, 1998; Naumann et al., 1994). For each experiment, lipid monolayers were prepared in the LE or LC phase, and the channel link was closed after compression. Reflectivity curves were recorded first for the pure lipid monolayer without protein in the subphase. After this, up to 300 µl cytochrome c solution was injected into the subphase through submersed injection holes located in the edge of the inner protein compartment. After 40 min. incubation time, NR measurements were performed. The final cytochrome c concentration was c₀ = 420 ± 30 nM in all experiments but for 50:50 DPPC-d62:DMPG-d54 in LC phase. Throughout the whole experiment, the lateral pressure  was recorded to ensure that equilibrium conditions were reached before the commencement of NR measurements. To aid data analysis of NR measurements on a D₂O subphase, samples of the subphase were collected during the experiments and their isotopic purity checked by proton nuclear magnetic resonance, which accurately quantified any changes in subphase neutron scattering length due to unavoidable atmospheric H-D exchange.

To achieve the highest possible precision in the quantification of the amount of adsorbed protein, mixtures of lipids with their alky chain perdeuterated analogs were used. This allowed the optimization of the scattering length density (SLD) contrast between the lipid monolayer and the adsorbed cytochrome c monolayer. For an improved characterization of the lipid monolayer itself, additional NR data were gathered on monolayers of identical chemical composition but at different proportions of chain perdeuterated lipid analogs in the monolayer and on different subphases (D₂O and H₂O), thus enabling a thorough contrast variation.

IRRAS measurements were performed using a Perkin-Elmer Spectrum 2000 spectrometer with a liquid N₂-cooled MCT detector. For each spectrum, 512 interferograms were acquired at a resolution of 4 cm¹ (acquisition time ~9 min). The system was equipped with a user-modified Specac (LOT, Langenberg, Germany) external reflection unit and a home-built film balance. The angle of incidence was 28° with respect to the surface norm. To maintain a constant water-vapor content and temperature, the set-up was placed in a hermetically sealed and thermal insulated sample container. The change of water level in the trough due to evaporation during the experiment was found to be negligible. Spreading of the lipid monolayers and injection of the protein solution were achieved by operating through small holes without opening the sample container.

Measurements were done by switching between two troughs at regular intervals (every 10 min) at the beam position using a home-built trough shuttle system controlled by the acquisition computer. One trough equipped with the inner protein compartment contained the monolayer system under study (sample), whereas the other (reference) was filled with the pure subphase. The shuttle motion did not cause any additional changes of the lateral pressure in the sample trough during the experiment compared to a trough kept fixed for the same time. Reflection-absorbance (RA) spectra were generated from subsequent sample and reference measurements using GRAMS Version 3.01 software (Galactic Industries Corp., Salem, NH). Here, RA is defined as RA = log(R_S/R_R), where R_S and R_R represent the reflectance of the sample and of the reference compartment, respectively. Each experiment consisted of a first step of typically 5 RA spectra recorded for the pure lipid monolayer without protein in the subphase ("lipid spectra"). Afterwards, up to 154 µl cytochrome c solution were injected to the subphase and allowed to equilibrate. The final concentration in all experiments was c₀ = 420 ± 30 nM. Then, in a second step, the "protein spectra" were recorded over a total time of up to 10 h.

Because H₂O exhibits a strong absorbance in the amide I region (1700-1600 cm¹), it is unsuitable for quantitative comparisons. Therefore, all IRRAS experiments were conducted with D₂O as a subphase, which shows only weak absorption in the amide I region.

After the injection of protein solution into the subphase, spectral features of the amide I bands became visible in the RA spectra. To remove the contributions of the lipid monolayer and the bulk water in the amide I region and to improve the compensation of water vapor bands, difference spectra were calculated from the protein spectra and the lipid spectra (see Data Analysis section).

	DATA ANALYSIS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS DATA ANALYSIS RESULTS DISCUSSION CONCLUSION REFERENCES

NR data

NR data were analyzed by least square fitting of multilayer models to the reflectivity curves using the program MULF, which implements the optical matrix method (Born and Wolf, 1993). Each layer in the fit is characterized by its thickness d_j, its SLD _j, and a Gaussian roughness _j. In our case, all fits were performed with _j = 0.

To characterize the pure lipid monolayer in terms of a head-group and a chain region, a two-layer model (four-parameter fit) was used as previously described in detail (Naumann et al., 1994, 1995). To reduce ambiguity due to the four free parameters, additional constraints were used:

1. The head group thickness was kept constant for all experiments at d_Hd = 8.0 Å. This value was consistent with a reasonable structural interpretation of the data (see below) and is in agreement with previous data of binary lipid monolayers (Bayerl et al., 1990) and DPPC bilayers (Nagle and Wiener, 1988).

2. The area per lipid molecule A_{Lip, calc} calculated from the NR data fitting according to

(1)

(b_Ch, calculated scattering length of chain region; _Ch, fitted SLD of chain region; d_Ch, fitted thickness of the chain region) was required to agree with the area per lipid molecule A_Lip,ex obtained from the film balance measurements within a confidence limit of 8%. This rather wide error limit also accounts for possible uncertainties in b_Ch by mixing deuterated and protonated lipids. Errors of the model fits were determined from an analysis of the variation of the fit quality parameter ² (Naumann et al., 1994, 1995) for different model fits to the data. Error limits of the fit parameters were taken from those fits that caused an increase of ² by 10% from its minimum value.

The presence of the protein for the case of the monolayer with cytochrome c in the subphase was considered as a third layer (Naumann et al., 1996; Johnson et al., 1991) with the parameters d_Pr (thickness of protein layer) and _Ch (SLD of protein layer). In the fitting process of this three-layer system, the values obtained for the pure lipid layer were taken as initial values for the fits. Because no expansion of the lipid film could occur upon protein absorption due to the closure of the protein compartment, the same boundary conditions as for the pure lipid monolayer, i.e., d_Hd fixed and A_Lip,calc in agreement with A_Lip,ex, were applied, and error limits of the fit parameters were obtained as above.

Summing up, data were fit with three free parameters and one boundary condition for the pure lipid monolayer and with five free parameters and one boundary condition for the lipid monolayer plus adsorbed protein. As in the latter case, the total layer thickness increased to more than 5 nm, this procedure always led to unique solutions for the free parameters (Johnson et al., 1991; Brumm et al., 1994; Naumann et al., 1994).

From the fitting results the value _Pr, the fraction of protein in the protein layer,

(2)

was calculated. Here, _W is the SLD of the subphase and _Pr,0 is the SLD of the pure protein, considering that _Pr,0 is a function of _W owing to the exchange of labile protons in the protein.

A gradual decrease of the isotopic enrichment of the D₂O subphase by vapor exchange, as determined by high-resolution proton NMR, was considered in the data analysis. In the course of a typical NR experiment, _W decreased from its initial value of ^{(99.8% D2O)} = 6.36 · 10⁶ Ų by up to 0.4 · 10⁶ Ų. The correction introduced for _W altered the values for _Pr by less than 6%.

IRRAS data

Although the lipid monolayer exhibits no absorbance in the amide I region of the IRRAS, it can be shown by simulations of the IRRAS spectra using the optical multilayer model (Dluhy, 1986) that monolayers with different values of the real part n of their complex refractive index n = n_j + i_j lead to distinct features in the spectra under conditions that the subphase imaginary part  is nonvanishing. If these features arise in the amide I peak region of an adsorbed protein layer, its peak height will not solely depend on the amount of protein adsorbed but also on the refractive index n of the lipid monolayer. Because n depends on the monolayer density, which in turn is closely related to the area per lipid molecule, identical amounts of adsorbed protein would give rise to different amide I peak heights in the LE and the LC phase.

This behavior of the amide I band was corrected for by subtracting the spectrum of the pure lipid monolayer from that of the same monolayer (and at the same lateral pressure) with protein adsorbed. This kind of first-order correction worked satisfactorily for D₂O subphases only where  is sufficiently small (Bertie et al., 1989).

For each set of protein data, those difference spectra that showed the least disturbance by water vapor bands were selected for further analysis, resulting in the selection of typically 20 difference spectra calculated from 10 protein spectra. For each selected difference spectrum in the region between 2000 and 1500 cm¹, the peak height of the amide I band was obtained from fitting two Gaussians (corresponding to the absorption bands of amide I at 1650 cm¹ and a band attributed to vibrations of aspartic acid and arginine side chains (Flach et al., 1994; Chirgadze et al., 1975)) and a linear baseline. Peak heights were averaged over up to 18 difference spectra calculated with the same lipid spectrum.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS DATA ANALYSIS RESULTS DISCUSSION CONCLUSION REFERENCES

We studied monolayers of PC-PG mixtures that contained 30, 50, or 80% of the anionic lipid to evaluate the dependence of cytochrome c binding on the monolayer charge. To consider additionally the effect of lipid demixing on protein binding, we studied monolayers where the chain lengths of the two lipids mismatched (C14 chains for PG, C16 chains for PC) and compared them with systems without mismatch (C16 or C14 chains for both PC and PG). Further differences between the samples studied were due to isotopic substitutions of the lipid chains by their perdeuterated analogs. This enabled scattering contrast optimization with the aim of a high fidelity of the adsorbed protein quantification by NR. The exact compositions of all samples used in this work are listed in Table 1.

Anionic monolayers without proteins

The compression isotherms of all binary mixtures used are shown in Fig. 1 with the positions marked at which NR and IRRAS measurements were performed before and after protein addition to the subphase. The isotopic substitution of the lipids according to Table 1 caused only slight deviations from the isotherms shown. For example, the replacement of DMPG by DMPG-d54 caused a shift of the transition point toward higher pressure of <1 mN/m while retaining the shape of the isotherm (data not shown). Representative examples of NR curves without and with adsorbed protein are shown in Fig. 2.

View larger version (29K): [in this window] [in a new window]   FIGURE 1   Pressure-area isotherms (compression mode) of the binary PC/PG monolayers studied. The arrows indicate the positions at which NR and IRRAS measurements (circles) or IRRAS measurements only (squares) were performed before and after protein addition.

View larger version (17K): [in this window] [in a new window]   FIGURE 2   Neutron reflection curves of selected binary PC/PG monolayers before (circles) and after (squares) cytochrome c adsorption. The lines represent the best fits of 2-layer models (pure lipid monolayers, dotted lines) and of 3-layer models (after cytochrome c adsorption, solid lines). The inserts show the SLD profiles obtained from these models. A. 70:30 DPPC-d62:DMPG, LE phase. B. 50:50 DPPC-d62:DMPG-d54, LC phase. C, 20:80 DPPC:DPPG-d62, LC phase.

For the monolayers containing 30 and 50 mol% DMPG, both NR and IRRAS showed the expected changes upon the transition from the LE to the LC phase state: From the NR point of view, the transition caused an increase of the chain region thickness of d_Ch  4 Å in both D₂O and H₂O contrast (cf. Table 2). The absolute values of monolayer thickness and SLD as summarized in Table 2 compare well with those of previous publications (Maierhofer and Bayerl, 1998; Brumm et al., 1994; Naumann et al., 1994).

View this table: [in this window] [in a new window]   TABLE 2   Characterization of the pure lipid monolayers in terms of their chain and headgroup region thickness (dCh and dHd) and SLD (Ch and Hd) as derived from two-layer model fits of the NR data

The IRRAS technique allowed a simultaneous observation of the DPPC-d62 and the DMPG component upon the LE-LC transition. This phase transition caused a shift to lower wave numbers for the asymmetric C-D (_LE = 2127.5 ± 0.5 cm¹,  = 2.35 ± 0.45 cm¹) and C-H (_LE = 2926.0 ± 0.5 cm¹,  = 3.5 ± 0.6 cm¹) stretching mode. The observed shift took place over a pressure range that coincided with the coexistence region of the monolayer as represented in Fig. 1. Furthermore, the total shift in wavenumber compares well to that observed for phospholipid bilayers of similar composition at the fluid/gel transition (Reinl and Bayerl, 1993).

Protein adsorption from the subphase

Adsorption kinetics and layer structure

View this table: [in this window] [in a new window]   TABLE 3   Initial monolayer lateral pressure 0 and the pressure increase after the injection of cytochrome c to the subphase at a concentration of 420 ± 30 nM for all monolayer samples studied

Determination of the amount of bound cytochrome c

View larger version (24K): [in this window] [in a new window]   FIGURE 3   IRRAS difference spectra of the amide I region of all lipid monolayer samples studied after the adsorption of cytochrome c. A, 70:30 DPPC-d62:DMPG, LE phase; B, 70:30 DPPC-d62:DMPG; C, 70:30 DMPC:DMPG; D, 70:30 DPPC-d62:DMPG, LC phase; E, F, 50:50 DPPC-d62:DMPG, LE and LC phase; G, 20:80 DPPC:DPPG-d62.

View this table: [in this window] [in a new window]   TABLE 5   Height of the amide I absorbance peak of the infrared reflectance/absorbance spectra and protein coverage Pr for both phase states and for all monolayer samples studied

View larger version (10K): [in this window] [in a new window]   FIGURE 4   Amount of cytochrome c bound to the lipid monolayer in terms of the protein coverage Pr versus the proportion of anionic lipid in a DPPC/DMPG monolayer. Pr was determined from NR (circles) using Eq. 2 and from IRRAS measurements (squares). Open symbols represent the LE phase and full symbols the LC phase of the monolayer.

Effect of monolayer phase state and lipid chain mismatch

View larger version (14K): [in this window] [in a new window]   FIGURE 5   Intensity of the amide I band and protein coverage Pr as obtained from IRRAS measurements of binary lipid monolayer systems containing 30% anionic lipids for LE and LC phase and different constituent chain lengths: C16/C14: 70:30 DPPC-d62:DMPG; C16/C16: DPPC-d62:DPPG; C14/C14: DMPC/DMPG. Open symbols represent the LE phase and full symbols the LC phase of the monolayer.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS DATA ANALYSIS RESULTS DISCUSSION CONCLUSION REFERENCES

The most striking result of this study is the phase-state dependence of protein binding to monolayers for samples containing 30% anionic PG under chain mismatch conditions, although this effect was not observed for ideal mixing conditions or for higher (50%) PG content. The phase-state dependence of water-soluble protein binding is well established for lipid bilayers on a solid support and is used for bio-separation purposes (Loidl-Stahlhofen et al., 1996). It was demonstrated that the domain formation in the gel phase of bilayers due to non-ideal mixing of its neutral and charged lipid components is the dominant contribution to this effect (Käsbauer and Bayerl, 1999). The creation of such domains with diameters of <20 nm (Gliss et al., 1998) causes an increase of the local charge density of the bilayer, rendering the electrostatic binding of oppositely charged proteins stronger and the protein packing more dense (Käsbauer and Bayerl, 1999). Despite the 2-3 orders of magnitude larger size of monolayer domains compared to those in bilayers, our results obtained for the 70:30 DPPC-d62:DMPG samples strongly suggest that the dominating mechanism that controls the electrostatic coupling of proteins to the lipid surface is similar for bilayers and monolayers.

One could be tempted to argue that the increase of lipid packing density at the LE-LC transition may provide a sufficiently high increase of the surface charge density to cause a Coulomb attraction between the oppositely charged proteins and the lipid monolayer. However, the negative result of our experiment using anionic and zwitterionic lipids of identical (C₁₄) chain length (samples 70:30 DMPC:DMPG and 70:30 DPPC-d62:DPPG) does not support this surmise. On the contrary, our finding that the value of _PR obtained for the chain-matched sample in the LC phase (70:30 DPPC-d62:DPPG) is identical within the experimental error to that of the 70:30 DPPC-d62:DMPG sample in the LE phase (Table 5) clearly disproves the homogeneous packing argument. Besides that, a significant contribution of closer lipid packing to the protein binding is not expected from electrostatic theory. We had previously shown that the electrostatic potential increase connected with the change of lipid packing density between the two phases (assuming homogeneous mixing) is well below the thermal energy and thus negligible (Käsbauer and Bayerl, 1999).

Therefore, domain formation in the monolayer with increasing lateral pressure seems the most likely explanation. This process can give rise to a substantial enrichment of one lipid component within the domain at the expense of the other component and thus may cause a significant increase of local charge density over the domain. Our results suggest that a chain mismatch resulting in a non-ideality of the lipid mixing is crucial for a higher protein coupling to the LC phase monolayer. Because the 3.1-nm diameter of cytochrome c is very small compared to monolayer domain sizes, we can assume that many protein molecules couple to a single domain. A prerequisite is that the charge enrichment within the domain by partial demixing is sufficiently high to provide an attractive electrostatic potential energy at or above the thermal energy. Demixing can occur when the accumulation of like lipids as next neighbors is energetically favorable over an association between unlike lipids. The underlying force is the van-der-Waals interaction between the lipid hydrocarbon chains. Two major factors tend to prevent demixing: entropy and Coulomb repulsion between like-charged lipids. In an uncharged layer, demixing would take place up to the level where the gain in energy due to like lipid interaction would make up for the loss in mixing entropy. However, in a system containing charged lipids, the Coulomb repulsion will shift this level at the expense of like neighbor interactions, depending on the percentage of the charged species in the mixture. Consequently, the closer a binary mixture comes to equimolarity the more it will reduce its tendency for demixing in a similar way as the increase of the proportion of charged lipids does. These considerations may offer a rationale for the different binding characteristics of the DPPC monolayers containing 30% and 50% DMPG: to obtain a significant demixing upon the transition to the LC phase, much more entropy would be required for compensation at 50% than at 30% DMPG. Moreover, Coulomb repulsion would provide a hindrance for demixing at 50% DMPG compared to 30% DMPG. For these reasons it is not surprising that _Pr depends on the lipid monolayer phase state only for the system containing 30% DMPG and not for the one with 50%.

The results obtained with the 20:80 DPPC:DPPG-d62 sample show that, at 50% anionic lipid content, the saturation level of _Pr has not been reached. Because the protein coupling can be expected to be of a Langmuir-type adsorption, enhanced binding to a charge-enriched domain can well overcompensate for the weaker binding to the charge-depleted regions.

Thus, local charge enrichment is a plausible explanation for the enhanced coupling to the 70:30 DPPC:DMPG system in the LC phase in comparison to the LE phase and to 70:30 DPPC-d62:DPPG and 70:30 DMPC:DMPG samples where mixing is rather ideal. A similar saturation level of anionic lipids, above which the control of protein binding via the lipid phase state is dominated by an overall Coulomb attraction, was previously observed for the case of bilayers on a solid support (Käsbauer and Bayerl, 1999).

In spite of several similarities regarding the protein coupling to monolayers and bilayers, there are a number of distinguishing features that should be kept in mind. In bilayers, we observed a virtually complete unbinding of cytochrome c for fluid phase conditions (Käsbauer and Bayerl, 1999; Loidl-Stahlhofen et al., 1996), whereas LE phase monolayers showed merely a reduction of the bound protein fraction. Two reasons may account for these differences. One is that we used for monolayers a PG proportion which was at least a factor of 10 higher than that used in bilayers to obtain a readily detectable single layer of adsorbed protein. Second, the energy to detach the protein from the fluid monolayer surface was solely thermal in nature, whereas, for the case of bilayers, the flow of the aqueous bulk phase provided the dominating force for detachment. A further difference between monolayers and bilayers is that we determined for the former the amount of bound protein in each phase, i.e., after adjustment of the lateral pressure to the desired phase state, cytochrome c was added to the subphase and (after sufficient incubation) quantified by the measurement. In bilayers, cytochrome c was coupled to the gel phase bilayer, the amount of protein bound was quantified, then the bilayer was transferred to the fluid state by raising the temperature, and the amount of protein was quantified again. The reason for the procedure used for the monolayers is that the bound cytochrome c may stabilize the LC phase domains upon expansion to the LE state and thus prevent an effective protein detachment. Finally, the growth of domains in monolayers is rather continuous over a large pressure range above the critical point, resulting in a gradient of charge density from the domain center to the edge. In contrast, bilayer domains are exceedingly small, formed instantaneously at the transition to the gel phase, and grow in number rather than in size (Gliss et al., 1999). As a result, the composition of a single bilayer domain is most likely rather homogeneous over its diameter. Thus, the detachment of the protein upon the dispersion of the domain within the fluid bilayer will occur more rapidly than for the case of the large monolayer domain.

	CONCLUSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS DATA ANALYSIS RESULTS DISCUSSION CONCLUSION REFERENCES

The results of the work strongly suggest that the domain formation in lipid monolayers consisting of binary lipid mixtures of an anionic and a neutral lipid component can alter the amount of cytochrome c that binds to the monolayer. The driving force is the demixing of the two lipid constituents at the transition from the LE to the LC phase.

Furthermore, this study shows that IRRAS measurements using a Langmuir trough shuttle system for water-vapor band compensation represent a very sensitive method for the detection of protein binding to monolayers and its changes due to variation of lipid composition and lateral pressure. The IRRAS results agree well with those obtained by NR and the combination of both techniques where NR is used for a calibration of the IRRAS data can save a substantial amount of rare and expensive neutron beam time.