Surface Pressure Induced Structural Effects in Photosynthetic

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Surface Pressure Induced Structural Effects in Photosynthetic Reaction Center Langmuir-Blodgett Films Paolo Facci,*,†,‡ Victor Erokhin,§ Sergio Paddeu,† and Claudio Nicolini† Institute of Biophysics, University of Genova, Genova, Italy, and EL.B.A. Fundation, Genova, Italy Received August 5, 1997. In Final Form: October 24, 1997X The structural modifications of photosynthetic reaction centers from Rhodobacter sphaeroides arranged in Langmuir films and after deposition onto solid substrate have been investigated as well as the variations in protein orientation as a function of surface pressure. While at low surface pressure (15-25 mN/m) protein integrity appears to be affected by the action of the surface tension, as results from the film behavior at the air-water interface and from optical spectroscopy data. When the surface pressure (3045 mN/m) is increased, protein structure is much better preserved. Moreover, at those deposition pressures, the film is characterized not only by a higher surface density but also by an increased anisotropy, as revealed by polarized absorption spectroscopy and confirmed by optical ellipsometry. Measurements of water content desorption in deposited films by means of a gravimetric transducer have consistently confirmed the preservation of protein structure at higher surface pressures.

Introduction Protein films, being organized molecular architectures, are objects of detailed investigations toward the design and realization of new kinds of devices as well as for the basic understanding of new phenomena resulting from their dense molecular organization.1-3 Among the available techniques allowing the formation of protein thin films, the Langmuir-Blodgett (LB) technique4 seems to be the only which can provide densely packed films with molecular resolution in their thickness and, if necessary, organized superstructures, i.e., alternating layers of functional molecules.5 This technique has been successfully used for depositing films of different kinds of proteins such as antibodies,6-9 enzymes,10-14 cytochromes,15-17 bacterio-18,19 and bovine20 rhodopsins, and photosynthetic reaction centers (RCs) from different * Author to whom correspondence should be addressed. E-mail: [email protected]. † University of Genova. ‡ Present address: Department of Physics, University of Parma, Viale delle Scienze, 43100 Parma, Italy. § EL.B.A. Foundation. X Abstract published in Advance ACS Abstracts, December 15, 1997. (1) Lvov, Yu.; Erokhin, V.; Zaitsev, S. Biol. Membr. 1991, 4 (9), 1477. (2) Lvov, Yu.; Decher, G. Crystallogr. Rep. 1994, 39, 628. (3) Nicolini, C. In Molecular Manufacturing; Nicolini, C., Ed.; Plenum Press: New York, 1996; p 1. (4) Roberts, G. G. Langmuir-Blodgett Films; Plenum Press: New York, 1990. (5) Kuhn, H. Pure Appl. Chem. 1981, 53, 2105. (6) Erokhin, V.; Kayushina, R.; Lvov, Yu.; Feigin, L. Nuovo Cimento 1990, 12D, 1253. (7) Turko, I.; Pikuleva, I.; Erokhin, V. Biol. Membr. 1991, 4 (10), 1745. (8) Facci, P.; Erokhin, V.; Antolini, F.; Nicolini, C. Thin Solid Films 1994, 237, 19. (9) Tronin, A.; Dubrovsky, T.; Nicolini, C. Langmuir 1995, 11, 385. (10) Langmuir, I.; Schaefer, V. J. J. Am. Chem. Soc. 1938, 60, 1351. (11) Fromherz, P.; Marcheva, D. FEBS Lett. 1975, 49, 329. (12) Zhu, D. G.; Petty, M. C.; Ancelin, H.; Yarwood, J. Thin Solid Films 1989, 176, 151. (13) Fujiwara, I.; Ohnishi, M.; Seto, J. Langmuir 1992, 8, 2219. (14) Antolini, F.; Paddeu, S.; Nicolini, C. Langmuir 1995, 11, 2719. (15) Turko, I.; Krivosheev, A.; Chaschin, V. L. Biol. Membr. 1992, 9, 529. (16) Erokhin, V.; Vakula, S.; Nicolini, C. Thin Solid Films 1994, 238, 88.

bacteria.21-24 In most cases protein functional activity is retained in the films.25 Besides, several new properties, connected with the nature of the organized molecular assembly rather than with the specific features of the individual molecules, have been found. Among them, film anisotropy,24 mobility of protein molecules during the deposition,26 and increased thermal stability27 were reported in the literature. More specifically, RC from different photosynthetic bacteria have been organized in LB films to be studied in an environment similar to biological membranes.21-24 On the other hand, these films are considered as promising candidates for future optoelectronics development. For example, solid state photovoltaic cells have been described and realized.28,29 Objects of the present study are LB films of RC from Rhodobacter sphaeroides. This large (100 kDa) transmembrane protein is made of three subunits (L, M, and H) and is involved into the first stage of photosynthesis providing photoinduced transmembrane electron transfer (100% quantum yield) in photosynthetic bacteria.30 The RC crystal structure has been reported in the litera(17) Tazi, A.; Hotchandani, S.; Munger, G.; Leblanc, R. M. Thin Solid Films 1994, 247, 240. (18) Hwang, S. B.; Korenbrot, J. I.; Stoeckenius, W. J. Membr. Biol. 1977, 36, 115. (19) Sukhorukov, G.; Lobyshev, V.; Erokhin, V. Mol. Mater. 1992, 1, 91. (20) Maxia, L.; Pepe, M.; Radicchi, G.; Nicolini, C. Biophys. J. 1995, 69, 1440. (21) Alegria, G.; Dutton, P. L. Biochim. Biophys. Acta 1991, 1057, 239. (22) Alegria, G.; Dutton, P. L. Biochim. Biophys. Acta 1991, 1057, 258. (23) Erokhin, V.; Kayushina, R.; Dembo, A.; Sabo, J.; Knox, P.; Kononenko, A. Mol. Cryst. Liq. Cryst. 1992, 221, 1. (24) Facci, P.; Erokhin, V.; Nicolini, C. Thin Solid Films 1994, 243, 403. (25) Erokhin, V.; Facci, P.; Nicolini, C. Biosensors Bioelectronics 1995, 10, 25. (26) Facci, P.; Radicchi, G.; Erokhin, V.; Nicolini, C. Thin Solid Films 1995, 269, 85. (27) Nicolini, C.; Erokhin, V.; Antolini, F.; Catasti, P.; Facci, P. Biochim. Biophys. Acta 1993, 1158, 273. (28) Tamura, T.; Sato, A.; Ajiki, S.; Sugino, H.; Hara, M.; Miyake, J. Biochem. Bioenerg. 1991, 26, 117. (29) Yasuda, Y.; Sugino, H.; Toyotama, H.; Hirata, Y.; Hara, M.; Miyake, J. Biochemistry and Bioenerg. 1994, 34, 135.

S0743-7463(97)00883-4 CCC: $15.00 © 1998 American Chemical Society Published on Web 01/06/1998

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Figure 1. Location of the chromophore constituting the electron transfer chain inside the subunits of the RC molecule.

ture.31,32 The electron transfer chain formed by a dimer of bacteriochlorophyll (Bchl2), bacteriopheophytin (Bphe), and two quinones (Qa and Qb) is shown in Figure 1 along with the schematic subunit structure. In natural conditions Bchl2, the primary donor in the RC electron transfer chain, is reduced by a multiheme cytochrome attached at the periplasmatic side of the protein; cytochrome is lost during extraction from the membrane.30 The RC solution used for film formation contains protein molecules surrounded, in the hydrophobic region, by the detergent used for the extraction; i.e., the spreading solution is made of two components. In spite of the amount of work in the field of RC LB films, several questions remain unanswered so far, namely, the effect of surface tension on protein integrity and the role of surface pressure on the anisotropy of the resulting films. The aim of this work, therefore, is to study these aspects. As the RC LB films are rather amorphous, it is not possible the use direct structural analyses such as X-ray or electron diffraction; therefore, the study has required the exploitation of several different experimental techniques and conclusions have been drawn based on the concurrent contributions of each method. Materials and Methods RC Solution. Protein solution was obtained from the Department of Biology of the Moscow State University. RCs were extracted and purified (purity at least 95%33) according to the procedure described in ref 33 and supplied at a concentration of 4.3 mg/mL (estimated by measuring the optical density at 803 nm (803 ) 288 mmol-1 cm-1)) in 10 mM Na-P buffer, pH 7.2, and 0.05% lauryldimethylamine N-oxide (LDAO) ((CH3)(CH2)11NO(CH3)2). This sample was used without any further purification. Monolayer Formation at the Air-Water Interface and Its Deposition onto Solid Substrate. Films were formed in a commercial trough (MDT Co. Russia) (surface area, 10 × 24 cm2; volume, 200 mL) and transferred onto solid substrates according to the procedure described in ref 27. Water purified by a Milli-Q system up to a resistivity of 18.2 MΩ cm was used as subphase. Three different kinds of substrates have been used, (30) Branden, C.; Tooze, J. Introduction to Protein Structure; Garland Publishing Inc.: New York, 1991. (31) Allen, J. P.; Feher, G.; Yeates, T. O.; Komiya, H.; Rees, D. C. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 5730. (32) Yeates, T. O.; Komiya, H.; Rees, D. C.; Allen, J. P.; Feher, G. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 6438. (33) Okamura, M. Y.; Steiner, L. A.; Feher, G. Biochemistry 1974, 13 (7), 1394.

Facci et al. namely, silicon plates for ellipsometry, quartz slides for optical spectroscopies, and quartz resonators for water content desorption measurements. From 2 to 20 µL of RC solution was spread over the water surface for each experiment according to the target working surface pressure (15-45 mN/m) in order to have not less than 60% of the target pressure just after spreading. The monolayer behavior at the air-water interface was studied by compressing the film up to the target deposition pressure at a speed of 30 cm2/min. Subsequently, the barriers were opened to the initial position at the same speed, and the monolayer was compressed once more to the target surface pressure. This procedure results in the removal of excess LDAO molecules from the water surface27 as their hydrocarbon chains (11 CH2 groups) do not allow the formation of a stable monolayer.4 Therefore, usually, the second compression gives rise to a decreased surface area occupied by the monolayer;27 if instead the area increases, it means that some processes take place, such as the loss of native protein structure which causes a larger area per molecule. For quantifying this behavior, the difference between the surface area occupied by the monolayer at the target pressure after the first (Area1) and second (Area2) compressions, normalized to the first, was plotted as a function of the target pressure. The parameter indicating the normalized area variation is defined as NAV and can be expressed as

NAV )

Area1 - Area2 Area1

(1)

Spectrophotometry and Linearly Polarized Absorption Spectroscopy. Fifty monolayers of RCs was deposited onto quartz slides for spectrophotometry and linearly polarized spectroscopy. Measurements were carried out under standard conditions (22 °C, 60% RH) in the wavelength range 200-1000 nm in the case of normal beam incidence (randomly polarized light) and in the 600-900 nm range in the case of beam incidence at 30° (linearly polarized light) on a Jasco 7800 double beam spectrophotometer (Jasco Co., Japan) using 0.2 nm resolution. Linearly polarized absorption spectroscopy has been performed by means of an in-house made module which allows an angle of 30° to form between the film plane and the beam pathway. This geometry allows recovery of the absorption due to the light linearly polarized along the film plane and that orthogonal to it by superimposing linearly the absorbances corresponding to the different polarizations. In order to check the role of light scattering in the measurements of the deposited films, relative ratios of absorbance at 280 and 350 nm (peptide bonds and quinones) in solution and film were estimated and found coincident, thus, enabling us to neglect it. Circular Dichroism. Circular dichroism (CD) measurements were performed with a Jasco 710 spectropolarimeter (Jasco Co., Japan) under standard conditions (22 °C, 60% RH) in the wavelength range 180-300 nm on samples containing 50 monolayers. Experimental conditions were as follows: resolution, 0.5 nm; time constant, 4 s; scan speed, 50 nm/min; band width, 2 nm; sensitivity, 10 mdeg. The technique used in this work followed the suggestions of de Jongh et al.34 for estimating the anisotropy degree in the LB RC films by measuring the orientation of the transmembrane R-helices as a function of the deposition surface pressure. The difference between the absorbance bands at 207 and 223 nm, due to R-helix contributions, normalized to their sum, was calculated to investigate film anisotropy. This parameter has been defined as CD anisotropy (CDA). Optical Ellipsometry. Ellipsometric measurements were carried out with a PCSA null ellipsometer equipped with a HeHe (λ ) 632.8 nm) laser source on samples containing three and five RC monolayers deposited onto silicon plates. Data were processed according to the two-layer model,35 and the average (34) de Jongh, H. H. J.; Goormaghtigh, E.; Killian, J. A. Biochemistry 1994, 33, 14521. (35) Tronin, A.; Dubrovsky, T.; De Nitti, C.; Gussoni, A.; Erokhin, V.; Nicolini, C. Thin Solid Films 1994, 238, 127.

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monolayer thickness was measured as a function of the deposition surface pressure. Water Content Desorption Measurements. A piezoelectric quartz nanobalance has been used according to the Sauerbrey equation36 for studying water desorption from LB RC films deposited at different surface pressures. The method is sensitive to protein structural variations induced by increasing temperatures.16 In fact, exposing the sample to increasing temperatures will cause progressive evaporation of the water present in the film. However, the water which is intimately connected with the protein molecules, either bound or trapped inside the protein globule, will be released and will be desorbed only after the proteins have undergone heat-induced16 structural modifications. As suggested,16 the experimental data (desorbed mass versus temperature) can be effectively fitted by the following formula:

A -b(T-Ttrans)

1+e

(2)

where A accounts for the total amount of desorbed water, Ttrans is the temperature corresponding to the maximum dehydration rate, b accounts for the steepness of the desorption curve, and T represents temperature. The first derivative of eq 2 with respect to temperature is proportional to the water desorption rate, which is the parameter (physical dimensions, mass/ temperature) used for quantifying the phenomenon. Such a model was shown to be suitable to explain the difference in the dehydration from closely packed LB RC films and selforganized dry smears, pointing out this technique as an effective approach for estimating changes in higher order protein structures.16 Five monolayers of RC were deposited onto quartz resonators (resonance frequency 10 MHz) at seven different surface pressures in the range 15-45 mN/m. Variations in the resonance frequency were measured by a home-made driving circuit37 equipped with a commercial frequencimeter (Good Fellow model GFC-8010G). Samples were exposed to each temperature for 5 min in a commercial oven, thermalized at room temperature, and measured. All these steps were carried out in parallel for all the samples.

Results and Discussion The study of the RC film behavior at the air-water interface is very important in order to assess the issues about surface tension induced protein damage. Generally speaking, the unfolding energy for practically all the kinds of proteins is in the range 21-63 kJ/mol.38 Simple considerations on the effects of the surface tension of pure water (72 mN/m) on a model protein (shaped like a cylinder of 5 nm diameter and (1-2) × 10-5 mol‚bar-1 compressibility39) yield a value of the work of the surface tension on the protein structure which is of the same order of magnitude (about 40 kJ/mol) as the unfolding energy. This figure was estimated by evaluating the energy associated with the volume variation of a molecule of given parameters when surface tension action is compensated by that of the elastic force. This observation entails that the probability of surface tension induced protein unfolding is not negligible. Therefore, the role of surface pressure in preventing protein denaturation could appear to be a key one, as increasing the surface pressure can cause a decrease of surface tension to the extent of preserving protein integrity. Several attempts were carried out toward the aim of decreasing surface tension, e.g., spreading proteins encapsulated in reversed micelles. Short chain surfactant molecules, being unable to form a (36) Sauerbrey, G. Z. Z. Phys. 1964, 178, 457. (37) Facci, P.; Erokhin, V.; Nicolini, C. Thin Solid Films 1993, 230, 86. (38) Pace, C. N. Trends Biochem. Sci. 1990, 15, 14. (39) Freiberg, A.; Ellervee, A.; Kukk, P.; Lasisaar, A.; Tars, M.; Timpmann, K. Chem. Phys. Lett. 1993, 214, 10.

Figure 2. Stability of RC monolayers at the air-water interface as a function of the target surface pressure.

stable monolayer on the water surface, reduce however the surface tension, thus preventing denaturation of proteins (particularly cytochrome c) which would be unfolded if spread alone.16 Another approach reported in literature for preventing surface tension induced protein unfolding deals with protein adsorption to a preformed monolayer.40 Aiming to address this issue, monolayer stability at the air-water interface was studied. Figure 2 reports the results of the experiments performed on different RC monolayers as a function of the target pressure. As is clear, at lower surface pressures (15 and 20 mN/m) the area occupied by the monolayer after the second compression is larger than that after the first, i.e., NAV (see Materials and Methods) has a negative value. These results can be interpreted in the framework of protein unfolding, which causes an increase in the area per molecule. At higher surface pressures (30, 35, and 40 mN/m) instead, NAV is positive and rather constant. This behavior is understandable in terms of removal of the excess of LDAO from the water surface and the attainment of protein close packing. At 25 mN/m surface pressure, the behavior of the film is intermediate between the two above mentioned situations, suggesting that still partial unfolding takes place. In the case of the monolayer at 45 mN/m, on the other hand, the NAV value increases significantly, indicating that a collapse of the RC monolayer begins to be significant. In order to have a deeper insight into the phenomenon, further investigations were carried out on films deposited onto solid substrates as a wider set of powerful experimental probes can be applied in this case. Figure 3 shows the family of optical absorption spectra for RC films deposited at different surface pressures. The band at 855 nm corresponding to the absorbance of Bchl2 (blue shifted of 10 nm in comparison with the solution counterpart, as already reported in the literature21) practically disappears in the case of lower surface pressures (15 and 20 mN/m) while that at 803 nm, connected to Bchl, is partially affected. Quantitative analysis of these data are presented in Figure 4, where the ratios between the absorbances at 280 (proteins) versus 803 nm (Bchl) (dashed line) and 803 versus 855 nm (Bchl2) (solid line) are plotted as a function of the surface pressure of deposition. In the dashed curve, the point at 15 mN/m indicates that the amount of Bchl chromophore compared to that of total proteins is about three times less than that in the samples deposited at the other surface pressures. As this chromophore is buried inside L and M subunits in the native protein structure, see Figure 1, this difference suggests that Bchl, in the (40) Peters, J.; Fromherz, P. Biochim. Biophys. Acta 1975, 394, 111.

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Figure 5. Water desorption rates from five-layer RC samples deposited at different surface pressures as a function of temperature. Numbers near the curves represent the transition temperatures.

Figure 3. Optical absorption spectra of 50 layers of RCs deposited at different surface pressures. Deposition surface pressures from the bottom to the top are in the range 15-45 mN/m with 5 mN/m steps.

Figure 4. Ratios between absorbance at 280 and 803 nm (dashed line) as well as 803 and 855 nm (solid line) as a function of the deposition surface pressure.

sample at 15 mN/m, has likely undergone surface tension induced protein unfolding and has been released into the bulk of the subphase. The solid line, instead, displays that the relative amount of Bchl and Bchl2 has a smoother trend, tending to a plateau value of 2 for higher surface pressures. Considering the information coming from both these curves, apart from the situation at 15 mN/m, the effect of surface tension is likely the result of splitting the protein globule into subunits. In fact, while the dashed line indicates that the integrity of the subunit structure is preserved at least to the extent of preserving the Bchl from being released, the solid line suggests that at low surface pressures Bchl does not form dimers; i.e., Bchl special pairs (see Figure 1) are not mutually placed in a configuration suitable for eliciting the dimer absorption

band to appear. As Bchl special pairs are located at the interface of L and M subunits, these data indicate that the subunits were likely split by surface tension. A further confirmation of the ideas arising from the previous experiments comes from the analysis of the data of water desorption upon temperature for LB RC films deposited at different surface pressures. In Figure 5 the water desorption rates for the whole set of samples are presented. They show that the transition temperature (that at which the desorption process reaches its maximum) is 50 °C only for the sample deposited at 15 mN/m while, in all the other cases, it is scattered around 100 °C. The data corresponding to surface pressures from 20 to 45 mN/m are in agreement with others already reported, which demonstrate that maximum desorption rate corresponds to the loss of protein quaternary structure.16 Instead, in the case of the sample at 15 mN/m, the transition temperature, much lower, points out once more that the film is made of proteins which are strongly affected in their structural integrity. Considering the previous results on the structure of the individual molecules in the LB film upon surface pressure, it is possible to study the effect of the surface pressure on the structure of the film itself in order to determine if RC molecules, which have in native conditions a very special orientation,30 retain it and to which extent in artificial LB films. This kind of information is indeed very important as allows assessing, for instance, the preferential direction of the light-induced electric dipoles which characterizes the functional behavior of RC when the cytochrome is removed from the complex and no acceptors are available.24,41 The thickness of RC monolayer and the absorbance at 280 nm corresponding to protein density in the film were measured by means of optical ellipsometry and photometry, respectively. The results are shown in Figure 6 as a function of surface deposition pressure. The two curves display a similar trend, increasing their value with pressure. It is worth noting that the maximum thickness value, reached at 45 mN/m (solid line), corresponds well to the protein size in transmembrane direction.42 Lower values of surface pressure allow some empty spaces in the LB film causing a lower effective thickness as the technique averages over the area illuminated by the laser spot. (41) Lukashev, E. P.; Kononenko, A. A.; Noks, P. P.; Gaiduk, V. I.; Tseitlin, M. B.; Rubin, A. B.; Betskii, O. V. Dokl. Acad. Nauk SSSR 1991, 316-318, 56. (42) Erokhin, V.; Feigin, L.; Kayushina, R.; Lvov, Yu.; Kononenko, A.; Knox, P.; Zakharova, N. Stud. Biophys. 1987, 122, 231.

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Figure 6. Optical absorbance at 280 nm (dashed line) and RC monolayer thickness (solid line) as a function of the deposition surface pressure.

Figure 8. CD spectra of 50 monolayers of RC deposited at different surface pressures. Deposition surface pressures from the top to the bottom are in the range 15-45 mN/m with 5 mN/m steps. Inset represents the normalized difference of the intensities of the CD bands at 207 and 223 nm.

Figure 7. Absorbance of linearly polarized light in the film plane (thick line) and normal to it (thin line) for 50 monolayer RC films deposited at different surface pressures. Deposition surface pressures from the bottom to the top are in the range 15-45 mN/m with 5 mN/m steps. The spectra are shifted by a constant offset.

Protein anisotropy in the LB films was studied by means of linearly polarized absorption spectroscopy and CD measurements. The first technique is sensitive to the orientation of the chromophores in the layer,43 while CD provides information on the orientation of R-helices when used on immobilized samples such as films.34 Figure 7 reports the absorbances for light polarized in directions parallel and and orthogonal to the film plane in the case of films deposited at different surface pressures. (43) Cantor, C. R.; Schimmel, P. R. Biophysical Chemistry; W. H. Freeman and Co.: San Francisco, CA, 1980.

At 15 mN/m, consistent with the previous results, the signal is very weak and progressively increases with surface pressure. A surface pressure of 25 mN/m represents the boundary value between two different situations: at lower surface pressures the band of the dimer (855 nm) is practically absent and only in parallel polarization does it begin to appear at 25 mN/m; the band corresponding to the monomer (803 nm) shows a marked difference in the two polarizations; for orthogonal polarization, the band of Bphe (767 nm) is lower than the parallel counterpart. Instead, from 35 mN/m the situation varies. A pronounced dimer band appears in both polarizations; the intensity of the monomer bands in the two polarization becomes similar and that of the Bphe in the orthogonal polarization overcomes the planar one. These data, consistent with similar ones already reported in the literature,21 suggest that surface pressure induces a variation in the chromophore orientation. The analysis of the orientation of R-helices in immobilized transmembrane protein films can provide even more direct proof of the structural features of the layer. Besides, in RC molecules, transmembrane R-helices are arranged along the transmembrane direction with a divergence of 20-25°.30 As shown by de Jongh et al., in these cases the absorption bands at 207 and 223 nm are sensitive indicators of the preferential orientation of R-helices and, hence, of the RC molecules. The results obtained with CD are reported in Figure 8 for different values of deposition surface pressures. The inset shows the CDA (see Materials and Methods)

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parameter as a function of surface pressure. Once more, it is notable that at the lowest pressures (15 and 20 mN/ m) CDA has a negative value, indicating that the preferential orientation of the R-helix axes lays on the film plane or, in any case, is not orthogonal to the film plane. This feature is compatible also with the mentioned effects of the surface tension which can cause partial or total unfolding of the protein structure. At higher surface pressures CDA becomes positive, being already at a plateau starting from 30 mN/m and suggesting that the direction of the helix axis is now oriented normally to the film plane. The data at 25 mN/m correspond to an intermediate situation. Conclusions Considering the whole set of results obtained during this study, it is possible to suggest a model for the behavior of the protein molecules in the monolayer and their subsequent arrangement in the deposited film. At lower surface pressures (below 15 mN/m) the protein structure is strongly affected by the surface tension, resulting, likely, in a partial protein unfolding. Therefore, it is impossible to deal with intact RCs working below such a surface pressure value. The protein behavior above 20 mN/m is characterized by the coexistence of two different phases in the layer: intact proteins and, likely, split protein subunits which are present in different relative amounts. With an increase of the surface pressure, the relative amount of intact proteins increases.

Facci et al.

In this framework it is possible to establish a simplified model for the structure of the protein layer which involves intact and affected molecules (split into subunits and/or partially unfolded) in different relative amount according to the value of the deposition surface pressure. At higher surface pressure, the relative amount of split molecules is rather small and orientation of RC molecules in the film resembles that present in the native membranessRCs are arranged with their major axis normal to the film plane. In the case of lower surface pressures, instead, the relative amount of split and unfolded proteins is much higher, and therefore, the arrangement of the transmembrane helices in the LB film is mostly in the film plane or undefined. Of course, the real situation is more complicated due to the coexistence of these two phases as well as to the possible presence of residual detergent in the film. However, it is possible to conclude that increasing the surface pressure results in the transition from a situation characterized by a larger amount of affected molecules to one in which structural RC properties are much more preserved. Acknowledgment. This work is dedicated to the memory of Professor Alexander A. Kononenko tragically deceased in January 1996. The RC preparation used in this work was the last one supplied by the laboratory of Professor Kononenko. The authors are grateful to Mr. Andrea Rossi for technical assistance in figure drawing. LA970883G