protein membrane obtained from photosynthetic bacteria Rhodopseudomonas viridis deposited on glass substrates. Membrane structures with small domains ...
Thin Solid Films, 243 (1994) 455-458
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Atomic force microscopy studies of photosynthetic protein membrane Langmuir-Blodgett films H. Yamada National Institute for Advanced Interdisciplinary Research, 1-1-4 Higashi, Tsukuba 305 (Japan)
Y. Hirata and M. Hara National Institute for Bioscience and Human Technology, 1-1-4 Higashi, Tsukuba 305 (Japan)
J. Miyake National Institute for Advanced Interdisciplinary Research, 1-1-4 Higashi, Tsukuba 305 (Japan)
Abstract Atomic force microscopy (AFM) has been used to investigate Langmuir-Blodgett films of the photosynthetic protein membrane obtained from photosynthetic bacteria Rhodopseudomonas viridis deposited on glass substrates. Membrane structures with small domains composed of hexagonally packed photoreaction units (PRUs) were imaged. The average spacing between PRUs in the AFM images is approximately 12 nm, which is in good agreement with transmission electron microscopy data. Observation on the 100 nm scale reveals that the films consist of membrane fragments of diameter ranging from 0.1 to 1 ]am. The recently developed force modulation technique was also used to image the local mechanical properties of the samples. There was a large difference between the images of the protein and the substrate.
1. Introduction Transmembrane proteins in photosynthetic bacteria undergo light-induced charge separation [1]. Because of this property, these films are attractive as models for photosensitive molecular devices. Several attempts have been made to develop protein thin films for realizing such molecular devices. Structural analysis of the thin films is essential in order to control the protein orientation and the arrangement. Atomic force microscopy (AFM) [2] has successfully demonstrated the capacity to image organic materials with molecular resolution [3, 4]. Its more general application to biology is greatly anticipated. A F M has the advantage of requiring neither special environments nor special sample preparations, such as staining, while transmission electron microscopy (TEM), often used for observing biological samples, needs both. There are still some difficulties, however, in imaging biomolecules with A F M mainly due to the relatively large interaction forces between the atomic force microscope tip and the sample compared with the weak adhesion of the sample to the substrate. Thus more work on immobilization of the sample is required for A F M imaging. In this study, we investigated Langmuir-Blodgett (LB) films of photosynthetic protein membranes de-
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posited on glass substrates in air using an atomic force microscope of our own design. The chromatophore membranes were obtained from the photosynthetic bacteria Rhodopseudomonas viridis.
2. Experimental procedure
2.1. Preparation of chromatophore Rhodopseudomonas viridis (ATCC No. 19567) was cultured as described by Drews and Giesbricht [5]. Cells were collected and stored at - 8 0 °C until use. After the bacterial cells were broken in a French pressure cell at 1200kgfcm -2 pressure, cell debris was removed by centrifugation for 10 min at 5500g. Then the chromatophore was washed three times for several hours at 4 °C and suspended in 10 mM Tris-HC1 buffer (pH 8.2) containing 1 mM CaCI2. The resulting chromatophore suspension, which has an optical density of 138 at a wavelength of 1020 nm, was spread on pure water from a Milli-Q system (Millipore, USA) containing 1 mM CaCI2, 1 mM Tris-HC1 (pH 8.2) and 0.5 mM sodium ascorbate at 15 °C. After surface pressure-area isotherms were measured, the films were deposited using a Langmuir trough (KSV-3000, KSV Instrument Co., Finland). Chromatophore monolayers were transferred
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H. Yamada et al. / AFM of photosynthetic protein membrane LB films
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to hydrophilic glass plates (Matsunami cover glass; thickness, 0.2 mm) by the vertical deposition method under a controlled surface pressure of 30 m N m - ~. We have attempted to use Muscovite mica, which has an atomically flat surface, as a substrate in place of the glass substrate and found that the membranes do not lie flat and aggregate when deposited on the mica surface. The poor adhesion of the membranes to the mica may be related to the fact that the mica has a negatively charged surface.
2.2. Apparatus An atomic force microscope constructed by the present authors was used in this experiment. The deflection of the cantilever is measured using the optical lever method. Laser light reflected by a chromatic beam splitter (CBS) is focused onto a cantilever. The deflection of the reflected light is measured with a two-segment photodiode. Using the CBS to direct the light beam provides enough space to introduce a microscope objective above the cantilever. It can be lowered to within 11 mm above the cantilever, so that commercial objectives with high numerical apertures can be used. Use of the atomic force microscope combined with the conventional optical microscope allows us to position the probe precisely at a desired location on the sample and to align the laser beam to the lever. When the CBS is removed, the objective can be lowered to within 4 mm of the lever. In this experiment, microfabricated cantilevers with tetrahedral tips made of single-crystal Si were used [6]. The calculated spring constant is 0.6 N m -1 and the resonance frequency is 110 kHz.
3. Results and discussion
Chromatophore, the photosynthetic membrane, is composed of lipids and photoreaction units (PRUs) as shown in Fig. 1 [7, 8]. Each P R U has a reaction center surrounded by six chlorophyll groups (light-harvesting protein (LH)). Each reaction center has four different
k 3 phototeaetion unit (PRU)
6 nm Fig. 2. TEM image of the chromatophore membrane. The bar shows the magnification.
subunits: C, H, L and M [9]. The C and H subunits project from the lipid bilayer in opposite directions. The C subunit has a relative positive charge while the H subunit has a negative charge. The orientations of all PRUs within one membrane are the same. Although individual H subunits tend to face the air-water interface, the orientation of membranes is not defined in the case of the large membrane. The T E M image, as in Fig. 2, shows that chromatophore has a pseudohexagonally packed two-dimensional quasi-crystalline structure with a spacing of 10-12 nm. Figure 3 shows a 1200 nm x 1200 nm A F M image of a protein membrane monolayer consisting of domains with the size ranging from 0.1 to 1 gm. Each domain corresponds to a fragment of chromatophore. The average size and the variation in the fragments are in good agreement with the TEM observation in Fig. 2. The average height of the domain walls, which were formed when fragments were pressed against each other during
5nm
Fig. 1. The schematic diagram of the sideview of chromatophore. C indicates the cytochrome, H, M, and L indicate the H subunit, the M subunit and the L subunit of the reaction center, and LH indicates the light-harvesting chrolophyll proteins.
400nm Fig. 3. 1200 n m × 1200 nm A F M image of the protein membrane monolayer deposited on the glass substrate.
H. Yamada et al. / A F M of photosynthetic protein membrane LB films
(a)
(b) 80nm
(c)
30 nm
80nm
(d)
30 nm
Fig. 4. (a), (b) Close-up views of (a) region A and (b) region B in Fig. 3. Both regions are 240 n m x 240 nm. Each P R U can be seen as a small bright dot in both figures. (c), (d) A F M images of the square area in (a) taken during (c) forward scanning and (d) backward scanning. The scan area is 120 n m x 120 nm.
compression in the LB trough, is 10-13 nm with respect to the membrane surface. In some areas the glass substrate is exposed and, on the boundary of the membrane, a step structure was observed instead of a domain wall. The observed height of the step from the membrane to the exposed glass surface is about 12 nm corresponding to the thickness of the membrane. The surface roughness of the glass substrate was a few nanometers in a smaller area than 100nm x 100 nm although the height of the undulation in a larger region than about 1 lam x 1 ~tm exceeded about 50 nm. Figures 4(a) and 4(b) show close-up views of regions A and B respectively in Fig. 3. Both regions are 240 nm x 240 nm. Each PRU can be seen as a small bright dot in both figures. The average spacing of the PRUs is 12 nm, a value consistent with the spacing as obtained from the Fourier analysis of TEM data [7, 8]. The observed corrugation amplitude of proteins is about 1.4 nm. Figure 4(b) shows a regular hexagonally packed structure while a partially ordered structure is visible in Fig. 4(a). Most of the observed membranes have a similar structure to one of these two pictures. As already mentioned concerning Fig. 1, C subunits project from one side of the membrane while H subunits project from the other side. Consequently, after deposi-
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tion on the substrate, some membrane fragments have C subunits on the surfaces and the others have differing orientations of PRUs, i.e. H subunits on the surfaces. The difference in the observed packing structures might be ascribed to the orientations of the membranes. Since controlling the orientation of the membrane is greatly important, this difference is quite interesting. Figures 4(c) and 4(d) are A F M images of the square area in Fig. 4(a). Figure 4(c) was taken during the forward scan of the cantilever and Fig. 4(d) during the reverse scan. The two figures show similar details except for the structure around the edge of the pit in the center of each picture. The difference between the images of the two scan directions is attributed to the lateral force acting on the atomic force microscope tip. The lateral force comes from frictional force between the tip and the sample except for the steep structure. The overall similarity between the images indicates that friction does not play a large role in imaging and therefore the contrast is due mainly to topographical variations. Although each PRU can be resolved as shown in Fig. 4, subunits could not be seen even in the magnified images in this experiment. It is possible that the atomic force microscope tip could slightly move the protein molecules and/or deform them. A recently deveoped technique in A F M provides new information about the local mechanical properties of the surface using force modulation through a vibration of the vertical sample position while the atomic force microscope tip is in contact with the surface [ 10]. When the frequency of the vibration is higher than the cut-off frequency of the feedback circuit, this vibration of the sample causes vibration of the tip and variation in the forces applied to the sample. Because softer samples such as proteins are considered to have a lower stiffness than the substrate, the amplitude of the cantilever's motion is smaller and the phase is more delayed when the tip is on the protein. Thus the stiffness and/or the viscosity of the sample surface can be imaged by the amplitude and the phase of the cantilever's response. In this way, different materials with the same topographic features are considered to give different contrasts. In this experiment the sample was vibrated with an amplitude of 0.5 nm at a frequency of 25 kHz. Figure 5(a) is a normal A F M image showing topographic data and Fig. 5(b) is the image obtained simultaneously from the in-phase signal caused by the modulation. The darkest areas in the image correspond to the protein membrane and brighter areas appear to be the glass substrate. However, the area in the right-hand portion near the center of the topographic image in Fig. 5(a) appears to be the glass substrate while the same region in the modulation image (Fig. 5(b)) displays intermediate contrast. One possible explanation for this observation is that the area in question is covered with a thin layer
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during forward scanning are the same as the images taken during backward scanning, except near exceptionally steep features, friction does not affect the image and the contrast comes mainly from the topography. Finally, the force modulation technique was used to image the local mechanical properties and was able to give different contrast between proteins and the substrate.
(a)
Acknowledgments
(b) 200 nm
200 nm
Fig. 5. (a) 600 nm x 600 nm AFM topographic image of the chromatophore membrane and the exposed region of the glass substrate. (b) The image obtained simultaneously from the in-phase component of modulated signal.
of proteins or lipids which come from broken membranes and which cause intermediate contrast.
4. Conclusion We demonstrated that the atomic force microscope is capable of imaging PRUs in the chromatophore deposited on the glass by the LB technique and analyzing the structure of these photosynthetic membranes. The membrane sample was obtained from Rhodopseudomonas viridis. The film consists of small membranes with size ranging from 0.1 to 1 lam. The PRUs show a regular, hexagonally packed structure in some membranes while a partially ordered structure can be seen in other membranes. The average spacing between PRUs in AFM images is approximately 12 nm, in good agreement with the TEM data. Since AFM images taken
We would like to thank Kan Nakayama for his continuous support and Shinya Akamine for a helpful discussion.
References 1 N. K. Packham, P. Mueller and P. L. Dutton, Biochim. Biophys. Acta, 933 (1988) 70. 2 G. Binnig, C. F. Quate and Ch. Gerber, Phys. Rev. Lett., 56 (1986) 930. 3 E. Meyer, L. Howald, R. M. Overney, H. Heinzelmann, J. Frommer, H.-J. Gfintherodt, T. Wagner, H. Schier and S. Roth, Nature, 349 (1991) 398. 4 H. Yamada, S. Akamine and C. F. Quate, Ultramicroscopy, 42-44 (1992) 1044. 5 G. Drews and P. Giesbrecht, Bakt. Arch. Mikrobiol., 53 (1966) 255. 6 S. Akamine, R. C. Barrett and C. F. Quate, Appl. Phys. Lett., 57 (1991) 316. 7 K. R. Miller, Nature, 30(1982) 53. 8 W. Stark, W. Kiihlbrandt, I. Wildhaber, E. Wehrli and K. Miihlethaler, EMBO J., 3 (1984) 777. 9 J. Deisenhofer, O. Epp, K. Miki, R. Huber and H. Michel, J. Mol. Biol., 180 (1984) 385. 10 M. Radmacher, R. W. Tillmann, M. Fritz and H. E. Gaub, Science, 2257 (1992) 1900.
