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* Laboratory for Polymer Physics, Laboratory for the Structure and Function of Biological Membranes, Structural Biology and Bioinformatics Center, Free University of Brussels, Brussels, Belgium
Correspondence: Address reprint requests to Dr. E. Goormaghtigh, Laboratory of Structure and Function of Biological Membranes, Université Libre de Bruxelles, CP 206/2, Boulevard du Triomphe, B-1050 Brussels, Belgium. Tel.: 32-2-650-5386; Fax: 32-2-650-5382; E-mail: egoor{at}ulb.ac.be.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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), and it does not require specific labeling, although labeling with 13C allows the study of selected residues (Torres et al., 2000, 2001). One of the main interests of P-ATR FTIR spectroscopy is that it yields insight into the orientation of the membrane molecules including protein secondary structures. As such, the method could provide a wealth of information on the organization of protein submolecular domains associated with the membrane. Orientational order parameters can indeed be derived from the dichroic ratio of the protein amide I or amide II bands. Yet, the determination of secondary structure orientation is subject to uncertainties from several origins. For the clarity of the subsequent discussion, we consider below that the orientation of an 
-helix long symmetry axis with respect to the internal reflection element (IRE) surface normal is to be determined. The discussion, however, stands in the general case for any transition with uniaxial symmetry. RATR is the experimental ratio between the absorbance of one band recorded with the incident light polarized parallel to the incidence plane (A||) and the absorbance recorded with the perpendicular polarization (A
):
 | (1) |
 | (2) |
and 
are the time-averaged square electric field amplitudes of the evanescent wave in the film at the IRE/film interface (Goormaghtigh and Ruysschaert, 1990). Sexperimental contains the information on the mean orientation of the transition dipole and on the distribution of the dipole orientations at their mean value. From FTIR dichroism measurements, only the projection of the angular distribution on the second Legendre polynomials can be measured (Rothschild and Clark, 1979). The latter is simply referred to as the order parameter (S) in the literature. The measured order parameter S, denoted below Sexperimental, is directly obtained from infrared dichroic ratios (Rothschild and Clark, 1979; Fringeli and Günthard, 1981), reviewed in Goormaghtigh and Ruysschaert (1990), assuming an uniaxial symmetry (Rothschild and Clark, 1979; Goormaghtigh and Ruysschaert, 1990; Bechinger et al., 1999).
The measured order parameter S, denoted Sexperimental, obtained from RATR through Eq. 2 can be generally expressed as the product of several order parameters related to a set of nested, uniaxial symmetric distributions (Rothschild and Clark, 1979). In this condition,
 | (3) |
0, ß0, and 0 as represented on Fig. 1, by the contribution of the disorder characterized by the distribution of the angular values at their mean.
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which characterizes the transition (no disordering). The value Sdipole can therefore be calculated as (3 cos20–1)/2 and detailed discussions about the orientation 0 of the transition dipoles for different secondary structures have appeared (Axelsen et al., 1995; Citra and Axelsen, 1996; Marsh et al., 2000; Marsh, 2000; Pali and Marsh, 2001; Marsh and Pali, 2001). The value Shelix can then be evaluated from Sexperimental provided that Smembrane is known.
The ordering of the membrane on the ATR crystal is usually considered to be "good" but only a few publications indicate that Smembrane could be close to 1 (Rothschild et al., 1980; Zhang et al., 1995a,b). Polarized ATR spectra were recorded on the sarcoplasmic reticulum Ca2+-ATPase. The anisotropy was found to be the highest in dry films and was found to decrease upon increasing hydration and membrane thickness (Buchet et al., 1991). AFM was used for the first time to characterize the IRE surface in the presence and in the absence of a monomolecular film by Axelsen et al. (1995). On a 512 x 512 datapoint image covering a 2 x 2 µm region of the crystal, Axelsen determined an order parameter of 0.65 and 0.82 for untreated and silanized crystal respectively, suggesting that the acyl-silane chains effectively reduce surface imperfections. Yet, the problem of the ordering in thick membrane stacks remains largely unaddressed.
The case of the gastric ATPase-containing tubulovesicles is of particular interest. These vesicles are recycled from the plasma membrane into intracytoplasmic vesicles as a storage form for the gastric ATPase and can be extracted as such from the stomach apical cells; see Yao and Forte (2003) for a review. The main protein present in the membrane is the gastric H+,K+-ATPase, a glycosylated protein responsible for stomach lumen acidification. Importantly, it has been shown before that thick multilayers systems built by simple drying of a tubulovesicle suspension are stable for hours in an aqueous flow, indicating the presence of strong interactions between the membranes. It has also been shown in the same system that the enzyme remains active and fully accessible to ligands that shift its conformation from the E1 to the E2 form of the enzyme (Vander Stricht et al., 2001; Scheirlinckx et al., 2004), indicating that the multilayer stack also presents large aqueous channels in its structure that allow ligands to quickly migrate through an 
100-bilayers-thick stack. Such a case is not unique; membrane stacks containing biotinylated PE were also found to be fully accessible to streptavidin, i.e., a homotetrameric protein (4 x 13 kDa; Acha et al., 2001).
In the present communication we address the issue of the membrane ordering for stacks made out of tubulovesicle membranes of different thicknesses using atomic force microscopy. It is found that membrane ordering remains very good (Smembrane >0.9) up to the thickest stacks considered.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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). They were further purified from the microsomal fractions by centrifugation on a discontinuous sucrose density gradient at 100,000 g overnight. The material collected at the 8–30% sucrose interface is referred to as the tubulovesicles.
Protein assay
Proteins were assayed using the BCA kit from Pierce (Rockland, IL). Bovine serum albumin was used as standard.
ATPase activity
ATPase activity of the tubulovesicles was determined in a medium containing 40 mM HEPES, 2 mM ATP, 2 mM MgCl2 at pH 7.2 in the presence or in the absence of 20 mM KCl. The vesicles were incubated at 37°C for 15 min in this medium. The reaction was stopped by addition of 7% SDS (final concentration 1.75%). Inorganic phosphate formation was assayed according to Stanton (1968) except that the coloration was revealed with ascorbate. Sucrose was 8% (w/v) to keep iso-osmotic conditions. This is necessary to obtain sealed vesicles (Raussens et al., 1997). ATPase activity expressed in µmol h–1 per mg–1 protein at 37°C is 36 ± 7 and 111 ± 30 after addition of 14 µM nigericin for uncoupling conditions, in line with previously published values.
Techniques
FTIR spectroscopy
Attenuated total reflection infrared (ATR-FTIR) spectra were recorded on a Bruker Equinox 55 infrared spectrometer equipped with a liquid nitrogen cooled mercury-cadmium telluride detector. The internal reflection element (IRE) was a trapezoidal germanium ATR plate (50 x 20 x 2 mm) with an aperture angle of 45° yielding 25 internal reflections (ACM, Villier St. Frederic, France). 256 scans were averaged for each spectrum. Spectra were recorded at a nominal resolution of 2 cm–1. The spectrometer was continuously purged with air dried on a FTIR purge gas generator 75–62 Balston (Maidstone, England) at a flow rate of 10 l/min. Spectra were recorded with incident light polarized parallel or perpendicular relative to the incidence plane.
Atomic force microscopy
Atomic force microscopy (AFM) images were obtained using a multimode atomic force microscope coupled with a Nanoscope III controller (Digital Instruments, Santa Barbara, CA). The multimode atomic force microscope was equipped with an E-scanner. Samples were imaged in tapping mode with a rectangular silicon cantilever (Nanosensors, type NCH) in ambient conditions. Topographic, phase, and amplitude data were collected. The integral and proportional gains and the scan rate were optimized for providing the best correspondence between line trace and retrace. AFM data were not filtered, although the topographic image data were background-corrected to eliminate the sample tilt. The treatment of images and cross-section analysis were performed with a home-made software (Basire and Ivanov, 2000). Four different image sizes were used: 15 x 15 µm2 (1 datapoint every 38 nm), 5 x 5 µm2 (1 datapoint every 12 nm), 1 x 1 µm2 (1 datapoint every 2.5 nm), and 500 x 500 nm2 (1 datapoint every 1 nm).
Sample preparation
The germanium IRE were cleaned just before use sequentially with a lab detergent (Decontamin 11, SA InterSciences, Brussels, Belgium), distilled water, methanol, and chloroform. They were then placed in a plasma cleaner PDC-23G (Harrick, 1967) for 5 min. For ATR-FTIR, thin films were obtained as described by Fringeli and Günthard (1981) by slowly evaporating the tubulovesicles under a N2 stream on one side of a germanium IRE. The covered area was close to 3 cm2. Concentrations (lipid + protein) were computed assuming a protein/lipid ratio (w/w) of 1.2. Concentrations were adjusted to spread volumes between 5 and 15 µl over 3 cm2. For AFM imaging, 1-cm2 Ge crystal plates were cut from an ATR germanium trapezoidal plate with a diamond saw.
Analysis
P2
computation
The dichroic ratio RATR (Eqs. 1 and 2) does not only depend on the mean orientation of the transition dipole but also on the distribution of the values around the mean (Eq. 3). The orientation of the membranes with respect to the germanium surface can be characterized on the one hand by the mean value of their tilt with respect to the germanium surface and, on the other hand, by the disordering around this mean value. The latter is characterized by the shape of the distribution of the angular values at the mean. The mean membrane orientation is found here to be parallel to the germanium surface. This is in agreement with a uniaxial symmetry axis perpendicular to the surface of the germanium crystal and with the mode of preparation of the samples. Because of the geometry of the ATR experiment, the simplest and most efficient way to describe the distribution of the orientations is in terms of Legendre polynomials. In general, any distribution of the membrane patch tilts D(
) can be described using a series of Legendre polynomials Pn(cos) (Rothschild and Clark, 1979; Goormaghtigh and Ruysschaert, 1990),
 | (4) |
Pn values are the coefficients determined from the experimentally obtained orientation distribution. The odd terms of Pn are zero because of the symmetry with respect to the membrane plane (Rothschild and Clark, 1979). The first two even-order Legendre polynomials are given by
 | (5) |
; Goormaghtigh and Ruysschaert, 1990). The coefficient Pn can be evaluated as
 | (6) |
), of the distribution of the tilts at the mean value 0, the P0 and P2 coefficients fully describe the IR dichroism even if they probably poorly describe the bell-shaped angular distribution. The value of the coefficient P2 is usually called "order parameter," S, as it describes the disordering around the mean value for P-ATR experiments. In the present work, D() for the membranes is directly measured from AFM images and its projection on P2, i.e., P2 = Smembrane, evaluated according to Eq. 6. |
RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Atomic force microscopy
Images were recorded over 15 x 15 µm2, 5 x 5 µm2, 1 x 1 µm2, and 0.5 x 0.5 µm2. Typical amplitude and phase images are presented in Fig. 2 for 0, 0.47, and 4.7 µg/cm2 of tubulovesicle materials. Clearly, the clean germanium plate provided by the manufacturer presents grooves resulting from the polishing. Measurements at higher magnification indicate that the largest grooves are 50-nm wide (not shown). It must be noted that this is much smaller than the IR wave amplitude (2–5 µm in the range of interest for membrane studies). Remarkably, addition of tubulovesicle membranes in a small amount (approximately just enough to cover the area) smears out the amplitude image while the grooves resulting from the polishing can still be detected in the phase image. This result demonstrates that when small amounts of membranes are spread on the germanium area, a rather homogenous film is formed. Further addition of membrane materials fully hides these grooves.
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15–20 nm each. Fourier transform of several height distributions (but not all) revealed a periodicity 1/15–1/20 nm–1, a reasonable estimate for the overall thickness of the tubulovesicle membranes (not shown). This value is in agreement with the size of the sarcoplasmic Ca2+-ATPase, 14 nm (Toyoshima et al., 2000). The fact that steps are detected in height profiles and approximately correspond to a bilayer thickness indicates that the slopes reported below could be overestimated because these steps were considered as a membrane tilt.
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P2P2 was computed for all the profiles and images obtained in the course of this study (Eq. 6). At this stage, it was necessary to decide on which scale the slope must be evaluated. The slope to be considered in this work is the slope which characterizes the general lipid bilayer orientation but not the individual phospholipid molecules. Membrane-embedded protein orientation must, in polarized ATR-FTIR experiments, be defined with respect to the general membrane orientation to decide whether the tilt measured origins from the tilt of the protein secondary structure with respect to the membrane, or from the tilt of the membrane itself with respect to the germanium support. In turn, relevant slopes should be evaluated on a distance D slightly longer than the molecular size. A phospholipid molecule has a diameter 
0.85 nm. A membrane-embedded protein has a much larger diameter (the diameter of the gastric ATPase, which is, by far the dominant protein in these membranes, is 4–5 nm). We tested several distances (1, 2, 5, 20, 50, and 200 nm) to compute the slopes. The resulting P2 obtained for different image sizes and resolutions are reported in Fig. 6 as a function of the distance D. It appears from Fig. 6 that the order parameter is always >0.95 with only a small increase upon increasing the distance over which the slope is computed. It turns out that the distance D and the image resolution have little effect on the value of P2.
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P2 is reported in Fig. 7 for tubulovesicle amounts varying between 0 and 47 µg spread over the 1-cm2 area. The slopes were computed over a distance of 2 nm. It appears that P2 is always >0.9, even for the largest amount tested.
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(C=O). Globally the dichroism of this band is close to zero and can be used as a good approximation of a magic-angle transition dichroism (Bechinger et al., 1999; Goormaghtigh et al., 1999). A major contribution of the lipids also appears at 1468 cm–1 and can be assigned to 
(CH2). Two important protein bands are found in the spectral window that exists in the phospholipid spectrum between 1700 and 1500 cm–1. Amide I (1700–1600 cm–1) is the most intense absorption band of the polypeptides. Amide (C=O) has a predominant role in amide I, accounting for 70–85% of the potential energy. (C-N) follows with 10–20% of the potential energy and the C-CN deformation may account for 10% of the potential energy (Krimm and Bandekar, 1986). Amide I also contains some in-plane NH bending contribution which is mainly responsible for the downshifts in the amide I frequency on N-deuteration (Krimm and Bandekar, 1986). Amide I shape is conformationally sensitive and generally used for secondary structure determination (for reviews, see Goormaghtigh et al., 1994a,b; Arrondo and Goni, 1999). The evolution of the integrated intensity of amide I, amide II, and lipid esterC=O as a function of the amount of membranes spread over the given crystal area appears in Fig. 9. As a first approximation, the spectral intensity increases as k (1–e–2z/dp) (Fringeli, 1992) where z is the film thickness from the germanium interface, k is a constant that depends on the extinction coefficient and dp is the penetration depth, i.e., the depth at which the electric field intensity falls to 1/e of its value at the interface (see Goormaghtigh et al., 1999 for a review). Using a membrane thickness of 15 nm (as mentioned earlier, this value is in agreement with the size of the sarcoplasmic Ca2+-ATPase, 14 nm—Toyoshima et al., 2000—and with the AFM data above) to evaluate the total Smembrane stack thickness, curve-fitting with k (1–e–2z/dp) (Fig. 9) for the ester (C=O), amide I, and amide II, yields dp = 0.298, 0.267, and 0.273 µm, respectively. This demonstrates the consistency of the measurements, and is in no bad agreement with the theoretical value of 0.378 µm at 1730 cm–1, considering the refractive indices of 4.0, 1.44, and 1.0 for germanium, the membranes, and air, respectively (see Goormaghtigh et al., 1994a). Fig. 10 indicates that the dichroic ratio, i.e., the ratio A||/A, also depends on the film thickness. This is due to the well known difference in the penetration depth of the parallel and perpendicular polarizations and has been described in detail before (Harrick, 1967). Fig. 10 indicates that converting the measured dichroic ratios in Sexperimental (Eq. 2) requires the knowledge of the film thickness. It has been suggested earlier that the lipid (C=O) can be used to retrieve the apparent film thickness (Bechinger et al., 1999) which fully explains the lipid (C=O) dichroism evolution with the amount of materials present in the film. Furthermore, this apparent thickness includes any lack of accuracy on the refractive indices (Bechinger et al., 1999). It must be stressed here that the ester (C=O) dichroic ratio should reach a theoretical value of 2 for infinite thickness (see Eq. 36 in Goormaghtigh et al., 1999). With only 100 bilayers, the data presented on Fig. 10 do not reach this limit value.
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85%). Assuming also that only the transmembrane segments (10 transmembrane helices for the -subunit and 1 for the ß-subunit) have a net orientation among the 1324 amino-acid-residue-long protein and assuming an average length of 20 residues per transmembrane segment, it turns out that 16% of the polypeptide chain would contribute to the dichroism. As demonstrated earlier, in such a case (Raussens et al., 1997) the dichroic ratio of the oriented helices R is given by
 | (7) |
as described earlier (Rothschild and Clark, 1979; Fringeli and Günthard, 1981; Goormaghtigh and Ruysschaert, 1990). The tilt of the dipole with respect to the helix axis has been evaluated to be 38° for the -helix (Marsh et al., 2000). Sdipole can be evaluated as (3 cos2(38°)–1)/2 = 0.43. The present work indicates that we can confidently assign a value of 0.9 for Smembrane. Shelix can now be calculated. Shelix was calculated for all the thicknesses investigated here, taking into account the apparent film thickness derived from the lipid (C=O) as described earlier (Bechinger et al., 1999). Its values range between 0.55 and 0.71, corresponding to an average tilt between 26° and 33° for the helix axis with respect to the membrane normal. These values are in close agreement with the recently obtained structure of another P-type ATPase (Toyoshima et al., 2000; Toyoshima and Nomura, 2002). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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The microscopic nature of the slopes reported in Fig. 5 must also be discussed. Fig. 4 suggests that discrete steps are present at the surface of the membrane stacks. As already mentioned, such steps have been unduly considered here as contributing to the tilt of the membranes. It turns out that including the effect of these steps in the overall tilt of the membranes can only result in an underestimate Smembrane.
It must also be stressed here that the mean slopes computed under the assumption that the cross-sections are traced at random angles are different from the dihedral angles characterizing the tilt of the membranes with respect to the supporting germanium surface. This effect potentially overestimates the value of Smembrane. The relation between this dihedral angle and the mean angle obtained from the experimental measurements was computed (not shown). It was found that within the range of values discussed in this article, the mean angle obtained experimentally is a good approximation of the dihedral angle (maximum difference <10°). When evaluated in terms of P2, the overestimation for a Gaussian distribution with a full width at half-height of 20° is 0.36. We consider that this error is acceptable in view of the overall accuracy of the FTIR measurements.
The finding that a rather homogenous film is formed upon drying the vesicle suspension on the germanium crystal (Figs. 2 and 3) is important because both spectral intensity and dichroism depend on film thickness. In turn, the coexistence of clusters of materials at some specific spots with empty spaces around would yield different dichroism results and would be essentially useless for quantitative evaluation of protein secondary structure orientations.
Importantly, Fig. 4 also demonstrates that a structural reorganization occurs, from a vesicular state to a planar system as demonstrated for pure lipid vesicles elsewhere (Johnson et al., 2002).
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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FOOTNOTES |
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Submitted on February 23, 2004; accepted for publication May 3, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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