Near-IR Absorption and Fluorescence Spectra and AFM Observation

The subunit light-harvesting 1 (LH 1) complexes isolated from photosynthetic bacteria Rhodospirillum rubrum using n-octyl-β-glucoside were reassociat...
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Langmuir 2005, 21, 3069-3075

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Near-IR Absorption and Fluorescence Spectra and AFM Observation of the Light-Harvesting 1 Complex on a Mica Substrate Refolded from the Subunit Light-Harvesting 1 Complexes of Photosynthetic Bacteria Rhodospirillum rubrum Kouji Iida,*,† Jun-ichi Inagaki,‡ Kiyoshi Shinohara,‡ Yoshiharu Suemori,‡ Makiko Ogawa,‡ Takehisa Dewa,‡ and Mamoru Nango*,‡ Nagoya Municipal Industrial Research Institute, Atsuta-ku Rokuban, Nagoya 456-0058, Japan, and Nagoya Institute of Technology, Showa-ku Gokiso, Nagoya 466-8555, Japan Received October 15, 2004. In Final Form: January 4, 2005 The subunit light-harvesting 1 (LH 1) complexes isolated from photosynthetic bacteria Rhodospirillum rubrum using n-octyl-β-glucoside were reassociated and adsorbed on a mica substrate using spin-coat methods with the aim of using this LH complex in a nanodevice. The near-IR absorption and fluorescence spectra of the LH 1 complexes indicated that the LH 1 complex on the mica was stable, and efficient energy transfer from a carotenoid to a bacteriochlorophyll a was observed. Atomic force microscopy of the reassociated LH 1 complexes, under air, showed the expected ringlike structure. The outer and inner diameters of the ringlike structure of the LH 1 complex were approximately 30 and 8 nm, respectively, and the ringlike structure protruded by 0.2-0.6 nm.

Introduction Light energy is absorbed by purple bacterium lightharvesting (LH) complexes and is rapidly transferred to the reaction centers (RCs) in the photosynthetic membrane, and that light energy is efficiently used to drive chemical reactions.1 Figure 1 illustrates the schematic side view of models of the LH 1/RC complex and the subunit LH 1 complex of Rhodospirillum rubrum (R. rubrum).2-4 The LH 1 complex surrounds the RC and consists of two short LH 1 polypeptides (R and β) coordinated with bacteriochlorophyll a (BChl a) (Figure 1b). The R or β polypeptides (Figure 2) bind BChl a. Their histidine residues (0) are liganded to the central metal magnesium of the BChl a’s. Figure 2 shows the amino acid sequences of the LH polypeptides from Rhodopseudomonas acidophila, Rhodobacter sphaeroides, and R. rubrum.1 There are two types of antenna complexes: peripheral LH 2 complexes and the LH 1 complexes.1 The structure of the LH 2 complex of R. acidophila strain 10050 has been resolved to a resolution of 2.0 Å.5 This LH 2 complex consists of a ring of nine heterodimeric subunits. However, such high-resolution structure has not yet been deter* Corresponding author. K. Iida: e-mail, iida.kouji@ nmiri.city.nagoya.jp; tel, +81-52-654-9904; fax, +81-52-654-6788. M. Nango: e-mail, [email protected]; tel, +81-52-735-5226; fax: +81-52-735-5226. † Nagoya Municipal Industrial Research Institute. ‡ Nagoya Institute of Technology. (1) Ke, B. Photosynthesis; Govindjee, Ed.; Kluwer Academic Publishers: Dordrecht, 2001. (2) Roszak, A. w.; Howard, T. D.; Southall, J.; Gardiner, A.; Law, C. J.; Isaacs, N. W.; Cogdell, R. J. Science 2003, 302, 1969-1972. (3) The LH 1 complex from R. rubrum was modeled by Fujitsu, Medicinal CAChe version 5 based on the LH 2 complex from R. acidophila. (4) The protein data bank URL is http://www.rcsb.org/pdb/ (identification code: RC, 1AIJ; LH 2, 1KZU). (5) McDermott, G.; Prince, S. M.; Freer, A. A.; HawthornthwaiteLawless, A. M.; Papiz, M. Z.; Cogdell, R. J.; Isaacs, N. W. Nature (London) 1995, 374, 517-521.

mined for the LH 1 complex. There are, however, lowresolution projection structures produced by transmission electron microscopy (TEM)6 of two-dimensional (2D) crystals of the LH 1 complex and a 4.8 Å X-ray crystal structure of the LH 1/RC2. TEM analysis of the LH 1 complexes revealed two types of complex, showing complete circular complexes from R. rubrum6 (Figure 2b) or open and C-C dimer complexes from R. sphaeroides (Figure 2c).7 The open ring LH 1 complexes could enable the diffusion of quinones (Q in Figure 2) from the RC to transfer electrons to cytochrome bc1 complex. Recently, the crystal structure resolved at 4.8 Å resolution of the LH 1/RC complex from Rhodoseudomonas palustris has been reported.2 This showed the RC surrounded by an oval LH 1 complex that consisted of 15 pairs of transmembrane helical R and β polypeptides and their coordinated BChl’s. A complete closure of the RC by the LH 1 is prevented by a single transmembrane helix (Figure 2d, W). Recently, atomic force microscopy (AFM) has been used to visualize membrane proteins, such as the LH 2 complexes.15-18 Scheuring et al. observed the LH 1 complex as a minor component together with the major LH 2 (6) Karrasch, S.; Bullough, P.; Ghosh, R. EMBO J. 1995, 14, 631638. (7) Jungas, C.; Ranck, J.-L.; Joliot, P.; Vermeglio, A. EMBO J. 1999, 18, 534-542. (8) Alegria, G.; Dutton, P. L. Biochim. Biophys. Acta 1991, 1057, 239-257. (9) Hirata, Y.; Nukanobu, K.; Hara, M.; Asada, Y.; Miyake, J.; Fujihira, M. Chem. Lett. 1992, 2277-2280. (10) Fang, J. Y.; Gaul, D. F.; Chumanov, G.; Cotton, T. M. Langmuir 1995, 11, 4366-4370. (11) Iida, K.; Kiriyama, H.; Fukai, A.; Konings, W. N.; Nango, M. Langmuir 2001, 17, 2821-2827. (12) Iida, K.; Kashiwada, A.; Nango, M. Colloids Surf., A 2000, 169, 199-208. (13) (a) Ogawa, M.; Kanda, R.; Dewa, T.; Iida, K.; Nango, M. Chem. Lett. 2002, 466-467. (b) Ogawa, M.; Shinohara, K.; Nakamura, Y.; Suemori, Y.; Nagata, M.; Iida, K.; Gardiner, A. T.; Cogdell, R. J.; Nango, M. Chem. Lett. 2004, 33, 772-773.

10.1021/la047460g CCC: $30.25 © 2005 American Chemical Society Published on Web 03/02/2005

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Figure 1. The side view of (a) the LH 1/RC complex and (b) the subunit LH 1 complex model. (a) The RC is taken from the R. sphaeroides structure. The computer-generated image of the LH 1 complex of R. rubrum is based on the crystal structure of the LH 2 complex from R. acidophila. The amino acid residues of the subunit LH 1 complex were replaced by the amino acids of the LH 2 polypeptides to those of LH 1 polypeptides using Fujitsu Medicinal CAChe version 5 (Fujitsu). The EVKQESL of N-terminal of LH-β polypeptides are not shown due to the lack of the corresponding LH 2 complex amino acid sequences. (b) The height length of the subunit LH complex was calculated by the Medicinal CAChe.

Figure 2. (a) Amino acid sequences of the LH 2 complex from R. acidophila and the LH 1 complex from R. rubrum and R. sphaeroides. (a) The histidine at 0 near the C-terminal is the binding site of the BChl a. The conserved amino acid residues are marked. Schematic drawing of the LH 1/RC complex of (b) R. rubrum, (c) R. sphaeroides, and (d) R. palustris.

complex from Rubrivivax geratinosus15 and the LH 1/RC complex in native photosynthetic membranes (chromato(14) Nagata, M.; Nango, M.; Kashiwada, A.; Yamada, S.; Ito, S.; Sawa, N.; Ogawa, M.; Iida, K.; Kurono, Y.; Ohtsuka, T. Chem. Lett. 2003, 32, 852. (15) Scheuring, S.; Reiss-Husson, F.; Engel, A.; Rigaud, J.-L.; Ranck, J.-L. EMBO J. 2001, 20, 3029-3035.

phore) from Rhodopseudomonas viridis16a and Rhodospirillum photometricum.16b Fotiadis et al. observed the LH 1/RC complex from R. rubrum in lipids from Escherichia (16) (a) Scheuring, S.; Seguin, J.; Marco, S.; Levy, D.; Bruno, R.; Rigaud, J.-L. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 1690-1693. (b) Scheuring, S.; Sturgis, J. N.; Prima, V.; Bernadac, A.; Le`vy, D.; Rigaud, J.-L. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 11293-11297.

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Figure 3. Procedure for the preparation of the LH 1 complexes from the photosynthetic membrane of R. rubrum and the production of a monolayer of the LH 1 complexes on the mica substrate. The LH 1/RC complexes in the photosynthetic membrane from R. rubrum were dissolved in an OG (30 mM) buffer (10 mM Tris, pH ) 7.5). The λmax of the LH 1 complexes shifted from 870 to 820 nm. The aqueous solution was applied to a Sephadex G-100 column (1.5 i.d. × 75 cm) to separate the RC and the subunit form of the LH 1 complex (B820). The subunit LH 1 complexes reassociated to the LH 1 complexes. The LH 1 complexes were spin-coated on freshly cleaved mica.

coli.17a Bahatyrova et al. showed that the LH 1 complexes of a mutant lacking the RC form circular, elliptical, and even polygonal ring shapes as well as arcs and open rings17b and that the LH 1 complexes are positioned to function as an energy collection hub from the LH 2 complex to RC in native membrane.17c Cryogenic AFM has also been useful for getting a high-resolution measurement of individual proteins in biological membranes, giving a strong boost to future studies in the field of soft material science.19 Scanning probe microscopy including AFM possesses the potential of not only imaging but also measuring a single biomolecule.20 Conductive AFM or scanning tunneling microscopy (STM) is also useful for studying the electron-transfer events. In this paper, we have studied the morphology and stability of LH 1 complexes refolded from the subunit LH 1 complex on mica by near-IR absorption and fluorescence spectra, as well as by AFM measurement. Further, the effect of carotenoids on the structure of the LH 1 complex on mica was examined. Figure 3 illustrates the procedures used in the purification of the LH 1 complex and its adsorption onto the mica surface. To remove RC from the LH 1/RC complex, the LH 1 complex is dissociated into the subunit LH 1 complexes (Figure 3a). It is known that the subunit LH 1 complex can be reassociated to the holoLH 1 complex which has a longer wavelength λmax near (17) (a) Fotiadis, D.; Quian, P.; Philippsen, A.; Bullough, P. A.; Engel, A.; Hunter, C. N. J. Biol. Chem. 2004, 279, 2063-2068. (b) Bahatyrova, S.; Frese, R. N.; van der Werf, K. O.; Otto, C.; Hunter, C. N.; Olsen, J. D. J. Biol. Chem. 2004, 279, 21327-21333. (c) Bahatyrova, S.; Frese, R. N.; Siebert, C. A.; Olsen, J. D.; van der Werf, K.; van Grondelle, R.; Niederman, R. A.; Bullogh, P. A.; Otto, C.; Hunter, C. N. Nature (London) 2004, 430, 1058-1062. (18) Stamouli, A.; Kafi, S.; Klein, D. C. G.; Oosterkamp, T. H.; Frenken, J. W. M.; Cogdell, R. J.; Aartsma, T. J. Biophys. J. 2003, 84, 2483-2491. (19) Yamamoto, D.; Tani, K.; Gotoh, T.; Kouyama, T. Micron 2003, 34, 9-18. (20) Lee, I.; Lee, W.; Greenbaum, E.; et al. Phys. Rev. Lett. 1997, 79, 3294.

873 nm, by decreasing the n-octyl-β-glucoside (OG) concentration (Figure 3b).21-23 However, the reassociated structure of the LH 1 complex is not yet known.21 Therefore, the LH 1 complex has been self-organized onto the mica surface in order to examine the structure of the complex by near-IR absorption and fluorescence spectra and by characterization with AFM (Figure 3c). The characterization of the self-organized LH 1 complex on the mica is expected not only to give important information but to help efforts to use LH complexes in a nanodevice.8-10,13-14 Experimental Section OG and Sephadex G-100 were purchased from Sigma Chemical Co. Growth of R. rubrum Wild-Type Bacterium. The photosynthetic bacterium R. rubrum wild type was grown anaerobically in modified Hutner’s media as previously described.21 Purification of the LH 1 Complexes. Chromatophores of R. rubrum were prepared as previously described.21 Carotenoid was extracted from chromatophores using benzene. Usually approximately 30 mM OG (1 mM Tris, pH 7.5) was added to dissolve chromatophores until the far-red absorption band shifted from 873 to 820 nm. Then the resultant aqueous solution was applied to a Sephadex G-100 column (1.5 cm i.d. × 75 cm) to separate the RC and LH 1 complexes with carotenoids and subunit LH/BChl a complexes without carotenoid (B820 complexes). The RCs were eluted immediately after the void volume and then the LH 1 complexes with carotenoid. Finally the B820 complexes were collected. λmax (absorbance) of the LH 1 complex in the absence of carotenoid (15 mM OG at 25 °C): 372 nm (0.86), 586 (0.23), 870 (1.14). λmax (absorbance) of the LH complex in (21) Visschers, R. W.; Chang, M. C.; van Mourik, F.; Parkes-Loach, P. S.; Heller, B. A.; Loach, P. A.; van Grondelle, R. Biochemistry 1991, 30, 5734-5742. (22) Miller, J. F.; Hinchigeri, S. B.; Parkes-Loach, P. S.; Callahan, P. M.; Sprinkle, J. R.; Riccobono, J. R.; Loach, P. A. Biochemistry 1987, 26, 5055-5062. (23) Chang, M. C.; Callahan, P. M.; Parkes-Loach, P. S.; Cotton, T. M.; Loach, P. A. Biochemistry 1990, 29, 421-429.

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the presence of carotenoid (20 mM OG at 25 °C): 372 nm (0.86), 475 (0.19), 512 (0.22), 546 (0.19), 586 (0.235), 875 (0.9). Near-IR and Fluorescence Spectra. Near-IR spectra were recorded with a Hitachi 3410. Fluorescence spectra were measured with a Nippon Roper fluorometer by using a halogen tungsten light bulb (TS-428 DC), a single monochromator (SP-150M) for selection of the excitation wavelength, a double monochromator (SP-306) and a CCD detector (Spec 10-100 BR/LN) to detect the emitted fluorescence. The slits were set at 0.50 mm for the LH 1 complexes in OG. Slits were set at 1.00 mm for the LH 1 complex on the mica. The solution samples were measured at 25 °C. The LH 1 complexes were spin-coated into the mica and rinsed by Milli-Q water. The measurements of LH 1 complex in the presence of carotenoid on the mica were made in 10 mM Tris pH 7.5 solution at 25 °C. AFM Measurement. The mica was freshly cleaved before every experiment using Scotch tape. A drop of sample (8-12 µM) was spin-coated on the mica and rinsed by Milli-Q water. The substrate was then dried. Imaging was performed with a commercial JSPM-4210 (JEOL). We used an AC Mode (Tapping Mode) and a standard silicon probe with either 7.5 N/m and 110 µm length (µMasch NSC 12) or 40 N/m and 125 µm length (Seiko Instruments Inc., SII). Computer-Generated Image Modeling. The procedure described here was carried out using the visualization program, Medicinal CAChe version 5 (Fujitsu Co.). The X-ray structure data of the LH 2 complex (Protein Data Bank (PDB): 1KZU) was opened by the CAChe. A LH 2 subunit complex (dimer) and corresponding BChl a’s were represented. The extra dimers and the carotenoids were removed for clarity. The corresponding amino acid residues were mutated to those of the LH 1 R and β polypeptides from R. rubrum by the mutation function of the CAChe to keep the backbone R helix of the LH 2 polypeptides, respectively. The configuration of the side chain of amino acid residues was not calculated, but just illustrated. The BChl a dimer (Figure 1b) is from the LH 2 complex. The RCs of R. sphaeroides (PDB: 1AIJ) and the resulting LH 1 dimer complexes (Figure 1b) were coupled to the LH 1/RC on the CAChe (Figure 1a). The relative height was adjusted to fit the X-ray crystallography.2

Results and Disscussion Near-IR Absorption and Fluorescence Spectra of the LH 1 Complexes on a Mica Substrate. It is now generally accepted that the subunit LH 1 complexes21 can be reassociated to give holo-LH 1 complexes where the BChl a Qy absorption is red shifted to 870 nm analogous to that seen from native LH 1 complexes (Figure 3b).12,22 The subunit LH 1 complexes are reassociated to the holoLH 1 complexes by dilution of the OG concentration below the critical micelle concentration (cmc) (20 mM).11-12, 21-23 The BChl a molecules then become excitonically aggregated, and the hydrophobic interactions between the subunit LH 1 complexes play an important role in the refolding process (Figure 3b).24 Linear dichroism (LD) of the LH 1 complex shows that the orientation of the Qy band of BChl a in this complex is perpendicular to the transmembrane axis (Figure 1b) as previously described.25 Figure 4 shows the absorption spectra of the reassociated LH 1 complex in the absence of carotenoids on a mica substrate and in OG micellar solution. It is apparent in Figure 4 that there is no 800 nm absorbance attributed to the BChl a of the RC, indicating that the RC was completely removed from the LH 1 complex. The Qy band of BChl a in the LH 1 complex on the mica was at 870 nm in the absence of carotenoids, which is consistent with that seen for the complex in aqueous solution at 25 °C. Similar results were obtained for the reassociated LH 1 (24) Iida, K.; Nango, M.; Yasue, H.; Okuda, K.; Okita, M.; Kashiwada, A.; Takada, N.; Maekawa, M.; Kurono, Y. Colloid Polym. Sci. 1998, 276, 152-159. (25) Iida, K.; Ohya, N.; Kashiwada, A.; Mimuro, M.; Nango, M. Bull. Chem. Soc. Jpn. 2000, 73, 221-229.

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Figure 4. Absorption spectra of the LH 1 complex in an OG micellar solution at 25 °C and spin-coated on mica at room temperature. The protein concentration was ∼8 µM. (Solid line) OG micellar solution; (dashed line) mica.

complex in the presence of carotenoids on the mica substrate. Table 1 shows the vis-near-IR absorption band of the LH 1 complexes. These results indicate that these LH 1 complexes were not denatured by binding to the mica surface. Similar results were obtained using a Langmuir-Blodgett (LB) film to lay down an LH 1 complex membrane layer on a glass substrate.12 Interestingly, the Qy absorbance value of BChl a in the LH 1 complex on the mica was similar to that of the monolayer LH 1 complex prepared by the LB method, implying that the LH 1 complex on the mica became a monolayer (Figure 3c). The LH 1 complex on the mica was stable for 24 h in a dark room at 4 °C. Figure 5a shows the fluorescence spectra of the reassociated LH 1 complex on the mica and in the OG micellar solution, when excited at 860 nm. Table 1 shows the fluorescence wavelength (λmax) of BChl a. The λmax of the spectra of the LH 1 complex on the mica was 5 nm redshifted to 895 nm in the presence of carotenoids compared to that in the absence of carotenoids. The fluorescence λmax of the spectra of the LH 1 complex both in the OG micellar solution and cast on the mica was 895 nm when excited at 510 nm. This indicated that an efficient energy transfer26 from carotenoid to BChl a in the LH 1 complex occurred both in the OG solution and on the mica. These data were consistent with those in the OG solution at 25 °C (Figure 5). The dissociation from the LH 1 complex in the presence of carotenoids to the subunit LH 1 complexes was more stable than that in the absence of carotenoids. Therefore, the carotenoid may have not only photofunction such as energy transfer but also improved structural stability, a useful property for the construction of monolayers. Oijen et al. successfully measured single molecule emission spectroscopy of the LH 2 complex spin-coated together with poly(vinyl alcohol) (PVA) on LiF.27 The AFM observation of the LH 2 complex on mica was reported by Schuering et al.15 In both cases the LH complexes were fully native. The absorption maxima of the LH 2 complex from R. acidophila on the mica were 802 and 858 nm, respectively, and the fluorescence maximum was 876 nm at room temperature (data not shown). These spectra were similar to those in OG micellar solution. Thus, these spectroscopic data indicate that the exciton states of the self-organized LH 1 and 2 complexes on the mica were also stable without denaturing as shown in Figure 3c. The AFM Images of the LH 1 Complexes in the Absence and Presence of Carotenoids. Figure 6 shows AFM images ((a) 1000 × 1000 nm2 and (b) 300 × 300 nm2) (26) Pullerits, T.; Visscher, K. J.; Hess, S.; Sundstro¨m, V.; Freiberg, A.; Timpman, K.; Grondelle, R. Biophys. J. 1994, 66, 236-248. (27) van Oijen, A.; Ketelaars, M.; Ko¨hler, J.; Aartsma, T. J.; Schmidt, J. Science 1999, 285, 400-402.

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Table 1. Near-IR Absorption Maxima (nm) and Fluorescence Maxima (nm) of the LH 1 Complex vis-near absorbance (nm) LH 1 complex (carotenoid)

OG aq or mica

LH 1 complex (-) LH 1 complex (-) LH 1 complex (+) LH 1 complex (+)

OG aq mica OG aq mica

carotenoid

BChl a Qy

475, 512, 546 475, 512, 546

870 870 875 875

fluorescence wavelength (nm) at 510 nm excitation

at 860 nm excitation

895 895a

890 890 895 895

a The emission of the LH 1 complexes by spin coating on mica was not observed in our equipment. This value was from the LH 1 complexes cast on mica.

Figure 5. Fluorescence spectra of the LH 1 complex on mica and in an OG micellar solution buffer at 4 °C. (a) The LH 1 complex in the absence of carotenoid was spin-coated. The excitation wavelength was 870 nm. (b) The LH 1 complex in the presence of carotenoid was cast. The excitation wavelength was 510 nm. The mica was in buffer solution. (Solid line) OG micellar solution; (dashed line) mica.

of LH 1 complexes in the absence of both RC and carotenoids on the mica. Ringlike structures were clearly observed in the 1000 × 1000 nm2 image (Figure 6a). The individual rings (Figure 6b, broken circle 1) and fused rings (Figure 6b, broken circle 2) could be seen. Figure 6c illustrates the line profile of Figure 6b (AA′-BB′). The diameters of the ringlike morphology were 29.0 (AA′) and 32.8 (BB′) nm for the outer surface and 8 nm for the inner surface, respectively. The average diameters of the ringlike structures were approximately 30 nm (outer) and 8 nm (inner) (n ) 24), respectively. The height of the rings was measured to be 0.2-0.6 nm above background (Figure 6c). These inner diameters are within the size for LH 1 rings predicted from X-ray and TEM studies.2,6 However, the outer diameter was too large (expected to be 12 nm). Scheuring et al. reported that the diameter between the ring center-top of the LH 1 complex without RC from R. gelatinosus is 10 nm.15 The outer diameter of the LH 1 complex/RC complex in the photosynthetic membrane from R. viridis16a and that in the reconstituted membrane from E. coli.17a are 12 and 16 nm (measured from printed material), respectively. In this study, the LH 1 complexes were transferred to the mica from OG detergent solution. Thus, our large outer diameter is likely to be due to OG

detergent remaining around the LH 1 complex, in which the OG molecules were adsorbed as approximately 5 layers (since the length of the OG molecules is approximately 2 nm). Although it is difficult to define the subunits at this level of resolution, the broken rings, which indicate the presence of incompletely refolded LH 1 complexes, were observed. LH 1 complexes where 30% of denatured LH 1 complexes were included (Figure 7a) and the subunit LH 1 complexes (Figure 7b), showed no ringlike structure. These data show that the formation of the ringlike morphology is due to the reassociated LH 1 complex structure and not from any artifacts such as adsorption of the OG detergent. Figure 8 shows the AFM images of LH 1 complexes in the presence of carotenoids, using an SII cantilever with 40 N/m. Interestingly, a 2D aggregated patch of LH 1 complexes was seen in the presence of carotenoids (Figures 8a and 6b), implying that the carotenoids may have a crucial role in the formation of those 2D aggregates. The height of the patch from the surrounding domain was 3-4 nm. The higher magnification images on the patch surface revealed roughly ringlike structure as shown in Figure 8c, although clearer ringlike structures were observed for the LH 1 complex in the absence of carotenoids (Figure 6). An inner diameter of 9 nm and an outer diameter of 36 nm were measured (n ) 20), and the height of the protruding LH 1 polypeptides was measured as 0.2-0.8 above the background. Fortiadist et al. show that the LH 1-RC complexes in E. coli lipid assemble in a lattice in aqueous solution.17 Stamouli et al. show that the LH 2 complexes in EggPC assemble in a lattice. 18 In this study, the LH 1 complexes from the subunit LH 1 complexes do not assemble in a lattice. This indicates that the LH 1 complex might randomly assemble in a monolayer in the presence of OG. It is important to immobilize these LH complexes on the various substrates without denaturation if these LH 1 complexes are to have reliable abilities for efficient energy transfer so that they can then be used for nonlinear optics or photosensitizers in a photonic nanodevice. The LH 1 complex with ZnBChl a, placed on an ITO electrode which had been modified with lipid bilayers, was stably assembled and showed photoinduced currents on the electrode.14 Karrash et al. reported that the detergent isolated LH 1 complex from R. rubrum formed a ringlike structure of the 16-mer of the subunit LH 1 complex.6 Recently, A. W. Roszak et al. reported the crystal structure at 4.8 Å resolution of the LH 1/RC complex from R. palustris and showed that the RCs are surrounded by an oval LH 1 complex.2 In this study, the carotenoid-less LH 1 complex was circular, not elliptical. This ringlike structure is in agreement with the detergent isolated LH 1 complexes visualized by TEM.6 This implies that the subunit LH 1 complexes alone without RC self-assemble into a complete circle. Thus, it may be that the specific interactions between RC and LH 1 complex deform the structure of the LH 1 complex.17a,28,29 However, Bahatyrova et al. show that the LH 1 complexes of a mutant lacking

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Figure 6. AFM images of the LH 1 complexes in the absence of both the RC and carotenoids reasssociated from the subunit LH complexes from R. rubrum on the mica. The cantilever was µMasch NSC 12, 7.5 N/m. (a) 1000 × 1000 nm2; (b) 300 × 300 nm2. The broken circles (1) denote the individual LH 1 complexes. The broken circles (2) denote the fused LH 1 complexes with OG detergent. (c) Height profile along the dashed white line of panel b.

Figure 7. AFM images of (a) the 30% denatured LH 1 complexes (500 × 500 nm2; vertical brightness range, 2.4 nm) and (b) the subunit LH 1 complexes (300 × 300 nm2; vertical brightness range, 3 nm). The cantilever was µMasch NSC 12, 7.5 N/m.

the RC from R. sphaeroides form circular, elliptical, and even polygonal ring shapes as well as arcs and open rings in the photosynthetic native membranes.17b The LH 1 complexes from R. sphaeroides form C-C dimers in the presence of RC in native photosynthetic membranes.17c,29

These results suggest that the LH 1 complex from R. sphaeroides is more flexible than those from R. palustris and R. rubrum. There are many polar amino acids such as glutamic acids (E) and asparagines (D) in the N-terminal region of the LH 1-R, β polypeptides from R. rubrum (Figure 2), which are out of the hydrophobic core as predicted by computer modeling in Figure 1b. This suggests that these amino acids at the N-terminal may be weakly adsorbed onto the mica surface as a result of hydrophilic interactions. Further, the C-terminal of the LH-R polypeptide also extends from the hydrophobic core according to the modeling scheme of Figure 1b. Scheuring et al. used proteolytic hydrolysis of the C-terminal region of the LH 2 complex from R. gelatinosus to show that the C-terminal surface of the LH 2 complex was oriented toward the AFM tip.15 Similar results were obtained by Stamouli et al.18 Thus, we can suggest that the N-terminal region of the LH 1 complex is oriented on the mica and the C-terminal region of the LH 1 complex is oriented toward the AFM tip (see Figures 1 and 3). The protruding height of the LH 1 complexes is predicted to be 1.8 nm from Figure 1b; however, the observed height in this study was 0.6 nm due to the adsorbed OG detergent. Although the tilt angle of the LH 1 complex by the LB method was 40° on a glass substrate which was roughly 20 nm (height) by 2 µm (area), the tilt angle might be close to 0° due to the atomically flat mica used in this study. This indicates that the Qy transition moment would be parallel to the mica surface. Thus, the LH 1 complex on the mica will be useful for studying the energy transfer between the LH 1 complexes or from carotenoids to BChl a in the LH 1 complex26 as well as for applying to energy transfer devices13,18 and nonlinear optics using the antenna function. Finally, the AFM technique could help in knowing the structure of the monolayer formed by self-assembly on mica. This shows that the sophistication of AFM observation will (28) We could not take the AFM image of the LH 1/RC, so far. Research to identify the high resolution of the LH 1/RC complex by AFM is being carried out. (29) Jamieson, S.; Wang, P.; Qian, P.; Kirkland, J. Y.; Conroy, M. J.; Hunter, C. N.; Bullough, P. A. EMBO J. 2002, 21, 3927-3935.

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Figure 8. AFM images of the LH 1 complexes in the presence of carotenoids from the reassociated subunit LH 1 complexes on mica. The cantilever was SII 40 N/m. (a) 1000 × 1000 nm2; vertical brightness range, 7 nm. (b) Illustration of panel a. (c) The LH 1 complex rich domain (square) of (a) 300 × 300 nm2. The broken circle (1) denotes the fused LH 1 complexes with OG detergent (see text). (d) Height profile along the dashed line of panel c.

help in a reasonable design of devices with the selforganized mixture of LH 2, LH 1, RC, and the LH model polypeptides.30 Conclusion We studied the structure of the two-dimensionally selforganized refolded LH 1 complexes on a mica substrate. The monolayer of the LH 1 complex on the mica was observed not to be denatured by the spectroscopic methods used. The AFM image of the LH 1 complexes, without RC and under air, shows ringlike structures which can be interpreted as individual molecules of the LH 1 complexes together until OG detergent. The dipole moments of the Qy band of BChl a complex in the LH 1 complex are horizontal on the mica, and an efficient energy transfer from carotenoid to BChl a could be seen. These results (30) Nango, M.; Kashiwada, A.; Watanabe, H.; Yamada, S.; Yamada, T.; Ogawa, M.; Tanaka, T.; Iida, K. Chem. Lett. 2002, 312-313.

demonstrate that the self-organization of the refolded LH 1 complexes on the mica can be a powerful tool to better understand the supramolecular structure of the LH 1 complexes as well as to gain knowledge about the functional conversion toward useful nanodevices. Acknowledgment. M.N. thanks Professors P. A. Loach and P. Parkes-Loach of Northwestern University for the kind gift of photosynthetic bacteria R. rubrum and Professor R. Cogdell of Glasgow University for helpful discussion. The present work was partially supported by NEDO International, Science, Tokyo, Japan, and by a Grant-in-Aid for Scientific Research on No. 13480186, 15033236, and 15655061 “priority area 417” from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of the Japanese Government, by the Tokyo Ohka Foundation for the Promotion of Science and Technology, and by the BBSRC Japan Partner Award. LA047460G