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Energy transfer and clustering of photosynthetic light-harvesting complexes in reconstituted lipid membranes
Chemical Physics 419 (2013) 200–204
Contents lists available at SciVerse ScienceDirect
Chemical Physics journal homepage: www.elsevier.com/locate/chemphys
Energy transfer and clustering of photosynthetic light-harvesting complexes in reconstituted lipid membranes Takehisa Dewa a,b,⇑, Ayumi Sumino a, Natsuko Watanabe a, Tomoyasu Noji a, Mamoru Nango a,⇑ a b
Department of Frontier Materials, Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan Japan Science and Technology, PRESTO, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan
a r t i c l e
i n f o
Article history: Available online 7 January 2013 Keywords: Photosynthetic light-harvesting complex LH2 LH1-RC Molecular assembly Energy transfer Reconstitution AFM
a b s t r a c t In purple photosynthetic bacteria, light-harvesting complex 2 (LH2) and light harvesting/reaction centre core complex (LH1-RC) play the key roles of capturing and transferring light energy and subsequent charge separation. These photosynthetic apparatuses form a supramolecular assembly; however, how the assembly influences the efficiency of energy conversion is not yet clear. We addressed this issue by evaluating the energy transfer in reconstituted photosynthetic protein complexes LH2 and LH1-RC and studying the structures and the membrane environment of the LH2/LH1-RC assemblies, which had been embedded into various lipid bilayers. Thus, LH2 and LH1-RC from Rhodopseudomonas palustris 2.1.6 were reconstituted in phosphatidylglycerol (PG), phosphatidylcholine (PC), and phosphatidylethanolamine (PE)/PG/cardiolipin (CL). Efficient energy transfer from LH2 to LH1-RC was observed in the PC and PE/PG/CL membranes. Atomic force microscopy revealed that LH2 and LH1-RC were heterogeneously distributed to form clusters in the PC and PE/PG/CL membranes. The results indicated that the phospholipid species influenced the cluster formation of LH2 and LH1-RC as well as the energy transfer efficiency. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction The light-harvesting (LH) complexes present in photosynthetic membranes are densely packed in lipid bilayers [1,2]. Such a clustered LH assembly is necessary for efficient light harvesting. In membranes of purple photosynthetic bacteria, light-harvesting complex 2 (LH2) and light-harvesting 1 (LH1)/reaction centre core complex (LH1-RC) are known to play important roles of capturing light and charge separation [3]. These photosynthetic apparatuses, composed of proteins and pigments (bacteriochlorophylls [BChls] and carotenoids [Car]), form a densely packed supramolecular assembly in the membrane. The LH2 in Rhodopseudomonas (Rps.) acidophila 10050 consists of 9 ab pairs of a-helical polypeptides, 27 BChla (9 B800 and 18 B850 chromophores), and 18 Car, forming a 9-membered cylindrical complex [4]. The LH1 in Rps. palustris possesses a 16-membered ellipsoidal structure consisting of 15 ab pairs of a-helical polypeptides, W polypeptide, 30 BChla (forming B880 chromophore), and Car. The LH1 is known to encircle the reaction centre complex (RC) to form a LH1-RC complex [5]. On absorbing light by B800 of LH2, an intramolecular energy transfer to B850 occurs in ⇑ Corresponding authors. Address: Department of Frontier Materials, Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan (T. Dewa). E-mail addresses: [email protected] (T. Dewa), [email protected] (M. Nango). 0301-0104/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chemphys.2012.12.039
1 ps, and generates excitonic 1B850⁄. The excitation energy is subsequently transferred between neighboring B850 of LH2 molecules [6] and further efficiently transferred to LH1 at 3–5 ps [7] and to RC at 35 ps [8,9], where charge separation takes place. Incidentally, if excess excited species (1B850⁄ and 3Car⁄) occur in the LH system, effective quenching mechanisms operate. These are referred to as singlet–singlet and singlet–triplet annihilations [10], and work during excitation energy transfers between B850 of LH2 complexes assembled into a densely packed cluster [11,12]. Recently, Pflock et al. reported that time-resolved fluorescence spectroscopy and dynamic Monte Carlo simulations for LH2 reconstituted into lipid bilayers, showing a relationship between energy transfer and annihilation [11,12]. Recently, atomic force microscopy (AFM) has revealed supramolecular organization of photosynthetic membranes, indicating the presence of polymorphic LH2/LH1-RC assemblies in photosynthetic bacteria [1,2,13]. Dimerized LH1-RC arrays are known to be connected to LH2 domains in Rhodobacter sphaeroides [1]. Likewise, hexagonally packed, paracrystalline clusters of LH2 and LH1-RC have been described in Rhodospirillum photometricum [2] and Rps. palustris [13], respectively. However, the role of such varied supramolecular organization in the function of the LH complexes remains unclear [14–17]. In addition, little is known about the effect of phospholipid molecules on such a LH2/LH1-RC assembly, its tendency to form clusters, packing density, and its miscibility in phospholipid bilayers. The understanding of lipid–protein
T. Dewa et al. / Chemical Physics 419 (2013) 200–204
interactions is important for the control of supramolecular architecture of photosynthetic membranes. However, no information on the effect of phospholipids on the supramolecular organization of photosynthetic membranes is available to date. In this study, we reconstituted LH2 and LH1-RC into various lipid bilayers to evaluate the energy transfers and the cluster formation of LH2 and LH1-RC in the membrane environment [15–18]. We demonstrated that phospholipids significantly influence the efficiency of energy transfer and the structure of molecular assembly consisting of LH2 and LH1-RC.
2. Materials and methods 2.1. Materials L-a-phosphatidylcholine (egg yolk; PC) and L-a-phosphatidylglycerol (egg yolk; PG) were gifted by Nippon Fine Chemical, Co., Ltd. L-a-phosphatidylethanolamine (E. coli; PE), PG (E. coli), and cardiolipin (E. coli; CL) were purchased from Avanti Polar Lipids, Inc. The phospholipids used in this study were PC (egg yolk), PG (egg yolk), and PE/PG/CL (E. coli) mixture (2/1/1, w/w/w), which mimics the bacterial photosynthetic membrane. The detergents used were n-octyl-b-D-glucopyranoside (OG) (Dojindo) and N, Ndimethyldodecylamine-N-oxide (LDAO) (Fluka). The photosynthetic membrane proteins, LH2 and LH1-RC were isolated from Rps. palustris 2.1.6, as previously described [5].
2.2. Reconstitution of LH complexes into lipid bilayers The phospholipids were dissolved in chloroform, and the solvent was evaporated under a nitrogen stream. The resulting lipid film was dried in vacuo for at least 6 h, after which it was hydrated in a Tris–HCl buffer (20 mM, pH 8.2) to furnish a multilamellar vesicle suspension. An OG solution was added to the suspension at a final concentration of 0.78 wt.% to give a co-micellar solution to which was added a LH-containing TL buffer (0.1 vol.% LDAO in 20 mM Tris–HCl buffer, pH 8.2) in a range of 50/1–5000/1 of the lipid/protein ratio (mol/mol). The detergent was removed by performing dialysis for at least 24 h at 4 °C [14–17]. For the calculation of the lipid/protein ratios, 1 CL molecule was considered as 2 molecules, since CL is a quasi-dimerized PG. The sucrose density gradient centrifugation showed that all LH complexes had been incorporated into liposomes. The structural integrity of LHs was examined by absorption spectroscopy before and after reconstitution.
2.3. Steady-state fluorescence spectroscopy LH-complexes-reconstituted liposomal solutions were subjected to steady-state fluorescence spectroscopy [15–17]. The concentration of LH2 in all sample solutions was adjusted to an optical density (O.D.) of 0.1 in the B850 absorption band. In the case of LH2/LH1-RC coexisting system, the absorption spectrum was deconvoluted into the individual spectra of LH2 and LH1-RC after which the O.D. of LH2 was adjusted to 0.1. Steady-state fluorescence spectra were obtained using a spectrometer equipped with a CCD detector (Spec-10: 100BR/LN; Roper Scientific), monochromators (SP-150 M for excitation and SP-306 for emission; Acton Research Co.), and a lamp house (tungsten halogen light source, TS-428DC; Acton Research Co.). All the data were obtained at room temperature, with excitation at either 800 nm (for LH2-only and LH2/LH1-RC existing systems) or 860 nm (for LH1-RC-only) at an exposure time of 4 s.
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2.4. AFM observation of reconstituted lipid bilayers A solution of reconstituted proteoliposomes, which were purified by sucrose density gradient centrifugation, was placed on a freshly cleaved mica surface followed by the addition of MgCl2 solution. The proteoliposomes were adsorbed on the mica surface via Mg2+, resulting in partially formed planar lipid bilayers [15,17]. After 1 h of incubation, the solution was gently replaced with Milli-Q water. The images were obtained with Picoplus5500 (Molecular Imaging) using AAC mode, at room temperature and cantilevers BL-AC40TS and AC-240TS (OLYMPUS) were used which had spring constants of approximately 0.1 and 2 N/m, respectively. Tip radii were <10 nm. Images were acquired under either aqueous (for PG and PC membranes) or ambient (for PE/PG/CL membrane) conditions.
3. Results and discussion 3.1. Absorption and fluorescence spectra of native photosynthetic and LH2/LH1-RC-reconstituted membranes The absorption and fluorescence spectra of suspensions of native photosynthetic membrane from Rps. palustris (Fig. 1A) showed 2 absorption bands characteristic of B800 (LH2) at 803 nm and B850 (LH2) + B880 (LH1-RC) at 860 nm, respectively. Further analysis of the peaks revealed that the latter band comprised LH2 and LH1-RC at a molar ratio of [LH2]/[LH1-RC] = 3.1. Upon excitation at 800 nm, a very broad emission was observed at 890 nm having a width of 56 nm (full width at half maximum, FWHM) (solid red lines). On adding a membrane-solublizing agent like LDAO, the absorption band (B850 + B880) blue-shifted by 4 nm (black dotted line) and the emission band (red dotted line) enhanced and blueshifted to 867 nm (41 nm of FWHM). As LH2 and LH1-RC are condensed in the native membrane, the absorption band red-shifted and the fluorescence from LH2 significantly quenched due to transfer of energy from LH2 to LH1-RC and the annihilation mechanisms [10–12]. The addition of LDAO relieved LH2 and LH1-RC from the condensed environment to enable emission mainly from LH2 upon excitation at 800 nm. Notably, similar spectral features (Fig. 1B) were observed on reconstituting LH2 and LH1-RC into PC membrane at a mol ratio of [LH2]/[LH1-RC] = 3.1 and a lipid/protein ratio of 250/1. The fluorescence quenching in the membrane was resolved by the addition of LDAO in a similar manner to that for the native membrane. Thus, the environment of the reconstituted membrane was spectroscopically similar to that of the native membrane. The spectral data of LH2 and LH1-RC in LDAO solution and the data on 3 types of phospholipid membranes have been summarized in Table 1. The absorption and emission bands were red-shifted in the reconstituted membranes compared to the LDAO-solubilized system. Further red-shift was observed on increasing the concentration in the membrane (from L/P = 500 to 50), which could be attributed to the modulation of pigment–pigment interaction between LH complexes. As mentioned above, fluorescence from the LH complexes was significantly quenched in the PC membrane. The fluorescence intensity plotted as a function of the protein/lipid ratio (Fig. 2) revealed that fluorescence intensity of both LH2 and LH1-RC significantly decreased upon increasing the protein/lipid ratio. This result suggested that with increasing the concentration the LH complexes in the membrane were condensed to form clusters. The relationship between the cluster size and excited singlet–singlet and singlet–triplet annihilations as the quenching mechanism has been reported [10–12]; with the increasing cluster size, the extent of annihilations are enhanced as a result of intercomplex energy
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Fig. 1. Absorption (solid black line) and fluorescence (solid red line) spectra of native photosynthetic membrane from Rps. palustris (A) and LH2/LH1-RC reconstituted into a PC membrane (B). Dotted spectra represent detergent-solubilized solution. (B), lipid/protein ratio = 250/1, [LH1-RC]/[LH2] = 1/3. Excitation wavelength was 800 nm. Spectra were acquired at room temperature.
Table 1 Spectral data of LH2 and LH1-RC in various environments. L/P Ratioa
a
c
LH1-RC (B880)
Absorption (nm)
Emissionb (nm)
Absorption (nm)
Emissionc (nm)
500 50
855 857 859
867 869 870
878 879 881
893 896 898
PC
500 50
857 859
869 870
879 881
896 898
PE/PG/ CL
500 50
857 859
869 870
879 881
896 898
LDAO PG
b
LH2 (B850)
Lipid/protein ratio (mol/mol). kex = 800 nm. kex = 860 nm.
transfer. Therefore, the formation of clusters led to fluorescence quenching. 3.2. Observation of energy transfer from LH2 to LH1-RC in various reconstituted membranes Fig. 3A shows the fluorescence spectrum of 1/1 mixture of LH2 and LH1-RC (Fig. 3A) (gray line) in a TL-buffer solution. Excitation wavelength was 800 nm, by which LH2 is selectively excited. The fluorescence profile was nearly identical to that obtained from LH2 (kem = 867 nm, black line (i)) and without the fluorescence component of LH1-RC (emission from LH1-RC by selective excitation at 860 nm as indicated by the black line (ii)). Thus, energy transfer from LH2 to LH1-RC was found to be negligible in the micellar solution since LH2 and LH1-RC are sufficiently away from each other. In contrast, the fluorescence band from the reconstituted membranes, PG (in B, blue line), PC (in C, red line), and PE/PG/CL (in D, green line) broadened upon irradiation at 800 nm. A clear emission band was observed at 869 nm upon irradiation at 800 nm (into the B800 band of LH2) from the membrane system of LH2 at L/P = 500 (Table 1). The membrane containing LH1-RC alone showed a very weak fluorescence at 896 nm upon excitation at 800 nm, as the LH1-RC does not absorb much at 800 nm [17]. The fluorescence band becomes prominent upon direct excitation to the B880 band of LH1-RC (Qy band of LH1 BChla, Table 1) [17]. Thus, the emission bands were significantly broadened (blue, red and green lines in Fig. 3) on LH2 and LH1-RC coexisting in the same membranes. Deconvolution of the spectra between fluorescence emission band
Fig. 2. Fluorescence intensity of reconstituted LH2 (circle) and LH1-RC (square) into PC lipid membranes as a function of protein/lipid ratio. Fluorescence intensity was normalized by the values at protein/lipid = 2 10 4.
of LH2 (solid black lines) and that of LH1-RC (dotted black lines) have been shown in B–D. The dissolved spectra clearly indicated that the emissions from LH2 were suppressed and those from LH1-RC were enhanced, which occurred due to an energy transfer from LH2 to LH1-RC. The ratios of the deconvoluted peak areas, ELH1-RC/ELH2, were 0.4 for PG (B), 1.7 for PC (C), and 1.7 for PE/PG/ CL (D). An enhanced energy transfer was observed in PC and PE/ PG/CL membranes in comparison to the PG membrane. AFM was used in order to investigate the relationship between the energy transfer efficiency and the morphology of LH2 and LH1-RC in the membranes. 3.3. Observation of the morphology of LH2/LH1-RC assembly in the lipid membranes The AFM images of LH2/LH1-RC-reconstituted membranes (A, PG; B, PC; and C, PE/PG/CL, Fig. 4) were acquired after placing a solution of reconstituted proteoliposomes on a mica surface, which led to formation of planar membranes by adsorption. In the PG membrane (A), lipid bilayer patches (4 nm in height) along with protrusions from the membrane surface (2–3 nm) were observed. While the protrusions represented the hydrophilic regions of LH2 and LH1-RC [15–17], they dispersed rather homogeneously in the PG membrane. A magnified image (A inset) revealed individual LH2 and LH1-RC complexes indicated by green and red arrows, respectively. The 2–4 nm protrusions in the PC membrane (B) surface were heterogeneously distributed to form clusters. The areas
T. Dewa et al. / Chemical Physics 419 (2013) 200–204
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Fig. 3. Fluorescence spectra of LH2/LH1-RC in a LDAO solution (A, gray line) and lipid membranes composed of PG (blue line in B), PC (red line in C), and PE/PG/CL (green line in D) membranes, respectively. Lipid/protein ratio was 500/1 (mol/mol). [LH1-RC]/[LH2] ratio was 1/1. Spectra of (i) and (ii) in (A) represent that of LH2-only (kex = 800 nm) and LH1-RC-only (kex = 860 nm), respectively. Solid and dotted spectra in B, C, and D represent dissolved spectra between fluorescence of LH2 (solid line) and LH1-RC (dotted line) obtained by waveform separation analysis. Excitation wavelength was 800 nm. Spectra were acquired at room temperature.
Fig. 4. AFM images of reconstituted LH2/LH1-RC assembly into lipid bilayers composed of PG (A), PC (B), and PE/PG/CL (C) membranes. Height profiles along the dotted line in the corresponding images were shown below their images. Insets in A and C: magnified images of the square areas within the gray and red lines in A and C, respectively. The areas encircled with dotted lines in B represent clusters of LH2/LH1-RC. Scale bars: 200 nm. Lipid/protein ratio was 500/1 (mol/mol). [LH1-RC]/[LH2] ratios were 1/4 in PG, 1/ 6 in PC, and 1/3 in PE/PG/CL membranes, respectively. Images were acquired under aqueous (A and B) and ambient (C) conditions, respectively. The PG membrane was prepared onto a poly L-lysine-modified mica as described in Ref. [16].
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encircled with dotted lines represent clusters of LH2/LH1-RC. At the resolution used, individual LH2 and LH1-RC were indistinguishable. Large particles (white spheres) in the image represented LH2/LH1-RC-containing proteoliposomes, which adhered to the surface and retained their spherical structures. This was probably due to the formation of protein-rich regions, which being stiff, prevented the transformation of the vesicles into a planar membrane structure. Thus, LH2 and LH1-RC dispersed heterogeneously to form a densely packed assembly in the PC membrane. Remarkably, the LH2 and LH1-RC in the PE/PG/CL membrane formed a densely packed assembly (C). A magnified image (C inset) revealed small and large protrusions indicated by green and red arrows representing LH2 and LH1-RC complexes, respectively. LH2 and LH1-RC distributed rather heterogeneously and homo pairing of LH1-RC was frequently found in the magnified image. In the native photosynthetic membrane, such LH1-RC homo pairing was often observed [2]. The distance between the paired LH1-RC complexes was determined to be 14.1 ± 1.5 nm (N = 38) in the PE/PG/CL membrane. As the diameter of LH1-RC was 11 nm, 3 nm of the space was occupied by the phospholipid molecules. Thus, the formation of such a densely packed LH2/LH1-RC assembly could possibly be ascribed to lipid–protein interactions. The AFM images clearly indicated that distribution and clustering of LH2 and LH1-RC depend on the phospholipid species, which may modulate the lipid–protein interactions to provide different types of protein clustering. It is plausible that bound lipids, e.g., PE and/or CL, are involved in the formation of densely packed assembly [19]. As mentioned above, the efficiency of energy transfer from LH2 to LH1-RC was found to be in the order of PE/PG/CL, PC > PG. Thus, the results of the morphology by AFM confirmed that densely packed and clustered LH2/LH1-RC assembly in the PC and PE/PG/ CL membranes exhibited more efficient energy transfer than in the PG membrane in which the LH2 and LH1-RC were homogeneously distributed. In a native photosynthetic membrane, as adjudged from the broadened emission, apparently an efficient energy transfer may take place as shown in Fig. 1A. The architecture of the LH complexes observed in the native membrane from Rps. palustris at the molecular level [13] showed that LH1-RC complexes were heterogeneously distributed to form hexagonally packed assemblies, and it would be interesting to study the influence of such an assembly on functions pertaining to energy transfers, charge separations, and subsequent electron/proton transport by quinones. Structural and functional analyses by AFM and other spectroscopies using reconstituted LH2/LH1-RC assembly may provide insights that aid in understanding the relationship between the structure of the antennae protein assembly and its functions. 4. Conclusion The relationship between the assembly of photosynthetic LH complexes and their function was addressed by reconstituting LH2
and LH1-RC into various lipid bilayers followed by an evaluation of the energy transfer and the structure of LH2/LH1-RC assembly. The efficiency of energy transfer from LH2 to LH1-RC was determined to be strongly dependant on the phospholipid species. AFM revealed that phospholipids influence the clustering of LH2 and LH1-RC in membrane environments and that the densely packed LH2/LH1-RC assembly exhibits an efficient energy transfer. Acknowledgments The authors are grateful to Prof. Dr. Richard J. Cogdell and Dr. Alastair T. Gardiner (University of Glasgow) for their kind gift of photosynthetic bacterial culture. This work was supported by PRESTO (Japan Science and Technology Agency, JST), CREST (JST), a Grant-in-Aid for Scientific Research on Priority Areas (477) No. 22018011 from the Ministry of Education, Culture, Sports, Science and Technology. MN and TD thank AOARD (124065) for funding. AS thanks the Grant-in-Aid for JSPS Fellows (23-02697) for fellowship. References [1] S. Bahatyrova, R.N. Frese, C.A. Siebert, J.D. Olsen, K.O. van der Werf, R. van Grondelle, R.A. Niederman, P.A. Bullough, C. Otto, C.N. Hunter, Nature 430 (2004) 1058. [2] S. Scheuring, J.N. Sturgis, Science 309 (2005) 484. [3] C.N. Hunter, F. Daldal, M.C. Thurnauer, J.T. Beatty (Eds.), The Photosynthetic Purple Bacteria, Springer, The Netherladns, 2009. [4] G. McDermott, S.M. Prince, A.A. Freer, A.M. Hawthornthwaite-Lawless, M.Z. Papiz, R.J. Cogdell, N.W. Isaacs, Nature 374 (1995) 517. [5] A.W. Roszak, T.D. Howard, J. Southall, A.T. Gardiner, C.J. Law, N.W. Isaacs, R.J. Cogdell, Science 302 (2003) 1969. [6] A.P. Shreve, J.K. Trautman, H.A. Frank, T.G. Owens, A.C. Albrecht, Biochim. Biophys. Acta 1058 (1991) 280. [7] S. Hess, M. Chachisvilis, K. Timpmann, M. Jones, G. Fowler, C. Hunter, V. Sundström, Proc. Natl. Acad. Sci. USA 92 (1995) 12333. [8] H. Bergström, R. van Grondelle, V. Sundström, FEBS Lett. 250 (1989) 503. [9] K.J. Visscher, H. Bergström, V. Sundström, C.N. Hunter, R. Grondelle, Photosynth. Res. 22 (1989) 211. [10] T.J. Pflock, S. Oellerich, J. Southall, R.J. Cogdell, G.M. Ullmann, J. Köhler, J. Phys. Chem. B 115 (2011) 8813. [11] T. Pflock, M. Dezi, G. Venturoli, R.J. Cogdell, J. Köhler, S. Oellerich, Photosynth. Res. 95 (2008) 291. [12] T.J. Pflock, S. Oellerich, L. Krapf, J. Southall, R.J. Cogdell, G.M. Ullmann, J. Köhler, J. Phys. Chem. B 115 (2011) 8821. [13] S. Scheuring, R.P. Gonçalves, P. Valérie, J.N. Sturgis, J. Mol. Biol. 358 (2006) 83. [14] T. Dewa, R. Sugiura, Y. Suemori, M. Sugimoto, T. Takeuchi, A. Hiro, K. Iida, A.T. Gardiner, R.J. Cogdell, M. Nango, Langmuir 22 (2006) 5412. [15] A. Sumino, T. Dewa, M. Kondo, T. Morii, H. Hashimoto, A.T. Gardiner, R.J. Cogdell, M. Nango, Langmuir 27 (2011) 1092. [16] A. Sumino, T. Dewa, T. Takeuchi, R. Sugiura, N. Sasaki, Biomacromolecules 12 (2011) 2850. [17] A. Sumino, T. Dewa, N. Sasaki, N. Watanabe, M. Kondo, T. Morii, H. Hashimoto, M. Nango, E-J. Surf. Sci. Nanotechnol. 9 (2011) 15. [18] A.R. Varga, L.A. Staehelin, J. Bacteriol. 161 (1985) 921. [19] N.J. Russell, J.K. Coleman, T.D. Howard, E. Johnston, R.J. Cogdell, Biochim. Biophys. Acta 1556 (2002) 247.
Contents lists available at SciVerse ScienceDirect
Chemical Physics journal homepage: www.elsevier.com/locate/chemphys
Energy transfer and clustering of photosynthetic light-harvesting complexes in reconstituted lipid membranes Takehisa Dewa a,b,⇑, Ayumi Sumino a, Natsuko Watanabe a, Tomoyasu Noji a, Mamoru Nango a,⇑ a b
Department of Frontier Materials, Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan Japan Science and Technology, PRESTO, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan
a r t i c l e
i n f o
Article history: Available online 7 January 2013 Keywords: Photosynthetic light-harvesting complex LH2 LH1-RC Molecular assembly Energy transfer Reconstitution AFM
a b s t r a c t In purple photosynthetic bacteria, light-harvesting complex 2 (LH2) and light harvesting/reaction centre core complex (LH1-RC) play the key roles of capturing and transferring light energy and subsequent charge separation. These photosynthetic apparatuses form a supramolecular assembly; however, how the assembly influences the efficiency of energy conversion is not yet clear. We addressed this issue by evaluating the energy transfer in reconstituted photosynthetic protein complexes LH2 and LH1-RC and studying the structures and the membrane environment of the LH2/LH1-RC assemblies, which had been embedded into various lipid bilayers. Thus, LH2 and LH1-RC from Rhodopseudomonas palustris 2.1.6 were reconstituted in phosphatidylglycerol (PG), phosphatidylcholine (PC), and phosphatidylethanolamine (PE)/PG/cardiolipin (CL). Efficient energy transfer from LH2 to LH1-RC was observed in the PC and PE/PG/CL membranes. Atomic force microscopy revealed that LH2 and LH1-RC were heterogeneously distributed to form clusters in the PC and PE/PG/CL membranes. The results indicated that the phospholipid species influenced the cluster formation of LH2 and LH1-RC as well as the energy transfer efficiency. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction The light-harvesting (LH) complexes present in photosynthetic membranes are densely packed in lipid bilayers [1,2]. Such a clustered LH assembly is necessary for efficient light harvesting. In membranes of purple photosynthetic bacteria, light-harvesting complex 2 (LH2) and light-harvesting 1 (LH1)/reaction centre core complex (LH1-RC) are known to play important roles of capturing light and charge separation [3]. These photosynthetic apparatuses, composed of proteins and pigments (bacteriochlorophylls [BChls] and carotenoids [Car]), form a densely packed supramolecular assembly in the membrane. The LH2 in Rhodopseudomonas (Rps.) acidophila 10050 consists of 9 ab pairs of a-helical polypeptides, 27 BChla (9 B800 and 18 B850 chromophores), and 18 Car, forming a 9-membered cylindrical complex [4]. The LH1 in Rps. palustris possesses a 16-membered ellipsoidal structure consisting of 15 ab pairs of a-helical polypeptides, W polypeptide, 30 BChla (forming B880 chromophore), and Car. The LH1 is known to encircle the reaction centre complex (RC) to form a LH1-RC complex [5]. On absorbing light by B800 of LH2, an intramolecular energy transfer to B850 occurs in ⇑ Corresponding authors. Address: Department of Frontier Materials, Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan (T. Dewa). E-mail addresses: [email protected] (T. Dewa), [email protected] (M. Nango). 0301-0104/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chemphys.2012.12.039
1 ps, and generates excitonic 1B850⁄. The excitation energy is subsequently transferred between neighboring B850 of LH2 molecules [6] and further efficiently transferred to LH1 at 3–5 ps [7] and to RC at 35 ps [8,9], where charge separation takes place. Incidentally, if excess excited species (1B850⁄ and 3Car⁄) occur in the LH system, effective quenching mechanisms operate. These are referred to as singlet–singlet and singlet–triplet annihilations [10], and work during excitation energy transfers between B850 of LH2 complexes assembled into a densely packed cluster [11,12]. Recently, Pflock et al. reported that time-resolved fluorescence spectroscopy and dynamic Monte Carlo simulations for LH2 reconstituted into lipid bilayers, showing a relationship between energy transfer and annihilation [11,12]. Recently, atomic force microscopy (AFM) has revealed supramolecular organization of photosynthetic membranes, indicating the presence of polymorphic LH2/LH1-RC assemblies in photosynthetic bacteria [1,2,13]. Dimerized LH1-RC arrays are known to be connected to LH2 domains in Rhodobacter sphaeroides [1]. Likewise, hexagonally packed, paracrystalline clusters of LH2 and LH1-RC have been described in Rhodospirillum photometricum [2] and Rps. palustris [13], respectively. However, the role of such varied supramolecular organization in the function of the LH complexes remains unclear [14–17]. In addition, little is known about the effect of phospholipid molecules on such a LH2/LH1-RC assembly, its tendency to form clusters, packing density, and its miscibility in phospholipid bilayers. The understanding of lipid–protein
T. Dewa et al. / Chemical Physics 419 (2013) 200–204
interactions is important for the control of supramolecular architecture of photosynthetic membranes. However, no information on the effect of phospholipids on the supramolecular organization of photosynthetic membranes is available to date. In this study, we reconstituted LH2 and LH1-RC into various lipid bilayers to evaluate the energy transfers and the cluster formation of LH2 and LH1-RC in the membrane environment [15–18]. We demonstrated that phospholipids significantly influence the efficiency of energy transfer and the structure of molecular assembly consisting of LH2 and LH1-RC.
2. Materials and methods 2.1. Materials L-a-phosphatidylcholine (egg yolk; PC) and L-a-phosphatidylglycerol (egg yolk; PG) were gifted by Nippon Fine Chemical, Co., Ltd. L-a-phosphatidylethanolamine (E. coli; PE), PG (E. coli), and cardiolipin (E. coli; CL) were purchased from Avanti Polar Lipids, Inc. The phospholipids used in this study were PC (egg yolk), PG (egg yolk), and PE/PG/CL (E. coli) mixture (2/1/1, w/w/w), which mimics the bacterial photosynthetic membrane. The detergents used were n-octyl-b-D-glucopyranoside (OG) (Dojindo) and N, Ndimethyldodecylamine-N-oxide (LDAO) (Fluka). The photosynthetic membrane proteins, LH2 and LH1-RC were isolated from Rps. palustris 2.1.6, as previously described [5].
2.2. Reconstitution of LH complexes into lipid bilayers The phospholipids were dissolved in chloroform, and the solvent was evaporated under a nitrogen stream. The resulting lipid film was dried in vacuo for at least 6 h, after which it was hydrated in a Tris–HCl buffer (20 mM, pH 8.2) to furnish a multilamellar vesicle suspension. An OG solution was added to the suspension at a final concentration of 0.78 wt.% to give a co-micellar solution to which was added a LH-containing TL buffer (0.1 vol.% LDAO in 20 mM Tris–HCl buffer, pH 8.2) in a range of 50/1–5000/1 of the lipid/protein ratio (mol/mol). The detergent was removed by performing dialysis for at least 24 h at 4 °C [14–17]. For the calculation of the lipid/protein ratios, 1 CL molecule was considered as 2 molecules, since CL is a quasi-dimerized PG. The sucrose density gradient centrifugation showed that all LH complexes had been incorporated into liposomes. The structural integrity of LHs was examined by absorption spectroscopy before and after reconstitution.
2.3. Steady-state fluorescence spectroscopy LH-complexes-reconstituted liposomal solutions were subjected to steady-state fluorescence spectroscopy [15–17]. The concentration of LH2 in all sample solutions was adjusted to an optical density (O.D.) of 0.1 in the B850 absorption band. In the case of LH2/LH1-RC coexisting system, the absorption spectrum was deconvoluted into the individual spectra of LH2 and LH1-RC after which the O.D. of LH2 was adjusted to 0.1. Steady-state fluorescence spectra were obtained using a spectrometer equipped with a CCD detector (Spec-10: 100BR/LN; Roper Scientific), monochromators (SP-150 M for excitation and SP-306 for emission; Acton Research Co.), and a lamp house (tungsten halogen light source, TS-428DC; Acton Research Co.). All the data were obtained at room temperature, with excitation at either 800 nm (for LH2-only and LH2/LH1-RC existing systems) or 860 nm (for LH1-RC-only) at an exposure time of 4 s.
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2.4. AFM observation of reconstituted lipid bilayers A solution of reconstituted proteoliposomes, which were purified by sucrose density gradient centrifugation, was placed on a freshly cleaved mica surface followed by the addition of MgCl2 solution. The proteoliposomes were adsorbed on the mica surface via Mg2+, resulting in partially formed planar lipid bilayers [15,17]. After 1 h of incubation, the solution was gently replaced with Milli-Q water. The images were obtained with Picoplus5500 (Molecular Imaging) using AAC mode, at room temperature and cantilevers BL-AC40TS and AC-240TS (OLYMPUS) were used which had spring constants of approximately 0.1 and 2 N/m, respectively. Tip radii were <10 nm. Images were acquired under either aqueous (for PG and PC membranes) or ambient (for PE/PG/CL membrane) conditions.
3. Results and discussion 3.1. Absorption and fluorescence spectra of native photosynthetic and LH2/LH1-RC-reconstituted membranes The absorption and fluorescence spectra of suspensions of native photosynthetic membrane from Rps. palustris (Fig. 1A) showed 2 absorption bands characteristic of B800 (LH2) at 803 nm and B850 (LH2) + B880 (LH1-RC) at 860 nm, respectively. Further analysis of the peaks revealed that the latter band comprised LH2 and LH1-RC at a molar ratio of [LH2]/[LH1-RC] = 3.1. Upon excitation at 800 nm, a very broad emission was observed at 890 nm having a width of 56 nm (full width at half maximum, FWHM) (solid red lines). On adding a membrane-solublizing agent like LDAO, the absorption band (B850 + B880) blue-shifted by 4 nm (black dotted line) and the emission band (red dotted line) enhanced and blueshifted to 867 nm (41 nm of FWHM). As LH2 and LH1-RC are condensed in the native membrane, the absorption band red-shifted and the fluorescence from LH2 significantly quenched due to transfer of energy from LH2 to LH1-RC and the annihilation mechanisms [10–12]. The addition of LDAO relieved LH2 and LH1-RC from the condensed environment to enable emission mainly from LH2 upon excitation at 800 nm. Notably, similar spectral features (Fig. 1B) were observed on reconstituting LH2 and LH1-RC into PC membrane at a mol ratio of [LH2]/[LH1-RC] = 3.1 and a lipid/protein ratio of 250/1. The fluorescence quenching in the membrane was resolved by the addition of LDAO in a similar manner to that for the native membrane. Thus, the environment of the reconstituted membrane was spectroscopically similar to that of the native membrane. The spectral data of LH2 and LH1-RC in LDAO solution and the data on 3 types of phospholipid membranes have been summarized in Table 1. The absorption and emission bands were red-shifted in the reconstituted membranes compared to the LDAO-solubilized system. Further red-shift was observed on increasing the concentration in the membrane (from L/P = 500 to 50), which could be attributed to the modulation of pigment–pigment interaction between LH complexes. As mentioned above, fluorescence from the LH complexes was significantly quenched in the PC membrane. The fluorescence intensity plotted as a function of the protein/lipid ratio (Fig. 2) revealed that fluorescence intensity of both LH2 and LH1-RC significantly decreased upon increasing the protein/lipid ratio. This result suggested that with increasing the concentration the LH complexes in the membrane were condensed to form clusters. The relationship between the cluster size and excited singlet–singlet and singlet–triplet annihilations as the quenching mechanism has been reported [10–12]; with the increasing cluster size, the extent of annihilations are enhanced as a result of intercomplex energy
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Fig. 1. Absorption (solid black line) and fluorescence (solid red line) spectra of native photosynthetic membrane from Rps. palustris (A) and LH2/LH1-RC reconstituted into a PC membrane (B). Dotted spectra represent detergent-solubilized solution. (B), lipid/protein ratio = 250/1, [LH1-RC]/[LH2] = 1/3. Excitation wavelength was 800 nm. Spectra were acquired at room temperature.
Table 1 Spectral data of LH2 and LH1-RC in various environments. L/P Ratioa
a
c
LH1-RC (B880)
Absorption (nm)
Emissionb (nm)
Absorption (nm)
Emissionc (nm)
500 50
855 857 859
867 869 870
878 879 881
893 896 898
PC
500 50
857 859
869 870
879 881
896 898
PE/PG/ CL
500 50
857 859
869 870
879 881
896 898
LDAO PG
b
LH2 (B850)
Lipid/protein ratio (mol/mol). kex = 800 nm. kex = 860 nm.
transfer. Therefore, the formation of clusters led to fluorescence quenching. 3.2. Observation of energy transfer from LH2 to LH1-RC in various reconstituted membranes Fig. 3A shows the fluorescence spectrum of 1/1 mixture of LH2 and LH1-RC (Fig. 3A) (gray line) in a TL-buffer solution. Excitation wavelength was 800 nm, by which LH2 is selectively excited. The fluorescence profile was nearly identical to that obtained from LH2 (kem = 867 nm, black line (i)) and without the fluorescence component of LH1-RC (emission from LH1-RC by selective excitation at 860 nm as indicated by the black line (ii)). Thus, energy transfer from LH2 to LH1-RC was found to be negligible in the micellar solution since LH2 and LH1-RC are sufficiently away from each other. In contrast, the fluorescence band from the reconstituted membranes, PG (in B, blue line), PC (in C, red line), and PE/PG/CL (in D, green line) broadened upon irradiation at 800 nm. A clear emission band was observed at 869 nm upon irradiation at 800 nm (into the B800 band of LH2) from the membrane system of LH2 at L/P = 500 (Table 1). The membrane containing LH1-RC alone showed a very weak fluorescence at 896 nm upon excitation at 800 nm, as the LH1-RC does not absorb much at 800 nm [17]. The fluorescence band becomes prominent upon direct excitation to the B880 band of LH1-RC (Qy band of LH1 BChla, Table 1) [17]. Thus, the emission bands were significantly broadened (blue, red and green lines in Fig. 3) on LH2 and LH1-RC coexisting in the same membranes. Deconvolution of the spectra between fluorescence emission band
Fig. 2. Fluorescence intensity of reconstituted LH2 (circle) and LH1-RC (square) into PC lipid membranes as a function of protein/lipid ratio. Fluorescence intensity was normalized by the values at protein/lipid = 2 10 4.
of LH2 (solid black lines) and that of LH1-RC (dotted black lines) have been shown in B–D. The dissolved spectra clearly indicated that the emissions from LH2 were suppressed and those from LH1-RC were enhanced, which occurred due to an energy transfer from LH2 to LH1-RC. The ratios of the deconvoluted peak areas, ELH1-RC/ELH2, were 0.4 for PG (B), 1.7 for PC (C), and 1.7 for PE/PG/ CL (D). An enhanced energy transfer was observed in PC and PE/ PG/CL membranes in comparison to the PG membrane. AFM was used in order to investigate the relationship between the energy transfer efficiency and the morphology of LH2 and LH1-RC in the membranes. 3.3. Observation of the morphology of LH2/LH1-RC assembly in the lipid membranes The AFM images of LH2/LH1-RC-reconstituted membranes (A, PG; B, PC; and C, PE/PG/CL, Fig. 4) were acquired after placing a solution of reconstituted proteoliposomes on a mica surface, which led to formation of planar membranes by adsorption. In the PG membrane (A), lipid bilayer patches (4 nm in height) along with protrusions from the membrane surface (2–3 nm) were observed. While the protrusions represented the hydrophilic regions of LH2 and LH1-RC [15–17], they dispersed rather homogeneously in the PG membrane. A magnified image (A inset) revealed individual LH2 and LH1-RC complexes indicated by green and red arrows, respectively. The 2–4 nm protrusions in the PC membrane (B) surface were heterogeneously distributed to form clusters. The areas
T. Dewa et al. / Chemical Physics 419 (2013) 200–204
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Fig. 3. Fluorescence spectra of LH2/LH1-RC in a LDAO solution (A, gray line) and lipid membranes composed of PG (blue line in B), PC (red line in C), and PE/PG/CL (green line in D) membranes, respectively. Lipid/protein ratio was 500/1 (mol/mol). [LH1-RC]/[LH2] ratio was 1/1. Spectra of (i) and (ii) in (A) represent that of LH2-only (kex = 800 nm) and LH1-RC-only (kex = 860 nm), respectively. Solid and dotted spectra in B, C, and D represent dissolved spectra between fluorescence of LH2 (solid line) and LH1-RC (dotted line) obtained by waveform separation analysis. Excitation wavelength was 800 nm. Spectra were acquired at room temperature.
Fig. 4. AFM images of reconstituted LH2/LH1-RC assembly into lipid bilayers composed of PG (A), PC (B), and PE/PG/CL (C) membranes. Height profiles along the dotted line in the corresponding images were shown below their images. Insets in A and C: magnified images of the square areas within the gray and red lines in A and C, respectively. The areas encircled with dotted lines in B represent clusters of LH2/LH1-RC. Scale bars: 200 nm. Lipid/protein ratio was 500/1 (mol/mol). [LH1-RC]/[LH2] ratios were 1/4 in PG, 1/ 6 in PC, and 1/3 in PE/PG/CL membranes, respectively. Images were acquired under aqueous (A and B) and ambient (C) conditions, respectively. The PG membrane was prepared onto a poly L-lysine-modified mica as described in Ref. [16].
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encircled with dotted lines represent clusters of LH2/LH1-RC. At the resolution used, individual LH2 and LH1-RC were indistinguishable. Large particles (white spheres) in the image represented LH2/LH1-RC-containing proteoliposomes, which adhered to the surface and retained their spherical structures. This was probably due to the formation of protein-rich regions, which being stiff, prevented the transformation of the vesicles into a planar membrane structure. Thus, LH2 and LH1-RC dispersed heterogeneously to form a densely packed assembly in the PC membrane. Remarkably, the LH2 and LH1-RC in the PE/PG/CL membrane formed a densely packed assembly (C). A magnified image (C inset) revealed small and large protrusions indicated by green and red arrows representing LH2 and LH1-RC complexes, respectively. LH2 and LH1-RC distributed rather heterogeneously and homo pairing of LH1-RC was frequently found in the magnified image. In the native photosynthetic membrane, such LH1-RC homo pairing was often observed [2]. The distance between the paired LH1-RC complexes was determined to be 14.1 ± 1.5 nm (N = 38) in the PE/PG/CL membrane. As the diameter of LH1-RC was 11 nm, 3 nm of the space was occupied by the phospholipid molecules. Thus, the formation of such a densely packed LH2/LH1-RC assembly could possibly be ascribed to lipid–protein interactions. The AFM images clearly indicated that distribution and clustering of LH2 and LH1-RC depend on the phospholipid species, which may modulate the lipid–protein interactions to provide different types of protein clustering. It is plausible that bound lipids, e.g., PE and/or CL, are involved in the formation of densely packed assembly [19]. As mentioned above, the efficiency of energy transfer from LH2 to LH1-RC was found to be in the order of PE/PG/CL, PC > PG. Thus, the results of the morphology by AFM confirmed that densely packed and clustered LH2/LH1-RC assembly in the PC and PE/PG/ CL membranes exhibited more efficient energy transfer than in the PG membrane in which the LH2 and LH1-RC were homogeneously distributed. In a native photosynthetic membrane, as adjudged from the broadened emission, apparently an efficient energy transfer may take place as shown in Fig. 1A. The architecture of the LH complexes observed in the native membrane from Rps. palustris at the molecular level [13] showed that LH1-RC complexes were heterogeneously distributed to form hexagonally packed assemblies, and it would be interesting to study the influence of such an assembly on functions pertaining to energy transfers, charge separations, and subsequent electron/proton transport by quinones. Structural and functional analyses by AFM and other spectroscopies using reconstituted LH2/LH1-RC assembly may provide insights that aid in understanding the relationship between the structure of the antennae protein assembly and its functions. 4. Conclusion The relationship between the assembly of photosynthetic LH complexes and their function was addressed by reconstituting LH2
and LH1-RC into various lipid bilayers followed by an evaluation of the energy transfer and the structure of LH2/LH1-RC assembly. The efficiency of energy transfer from LH2 to LH1-RC was determined to be strongly dependant on the phospholipid species. AFM revealed that phospholipids influence the clustering of LH2 and LH1-RC in membrane environments and that the densely packed LH2/LH1-RC assembly exhibits an efficient energy transfer. Acknowledgments The authors are grateful to Prof. Dr. Richard J. Cogdell and Dr. Alastair T. Gardiner (University of Glasgow) for their kind gift of photosynthetic bacterial culture. This work was supported by PRESTO (Japan Science and Technology Agency, JST), CREST (JST), a Grant-in-Aid for Scientific Research on Priority Areas (477) No. 22018011 from the Ministry of Education, Culture, Sports, Science and Technology. MN and TD thank AOARD (124065) for funding. AS thanks the Grant-in-Aid for JSPS Fellows (23-02697) for fellowship. References [1] S. Bahatyrova, R.N. Frese, C.A. Siebert, J.D. Olsen, K.O. van der Werf, R. van Grondelle, R.A. Niederman, P.A. Bullough, C. Otto, C.N. Hunter, Nature 430 (2004) 1058. [2] S. Scheuring, J.N. Sturgis, Science 309 (2005) 484. [3] C.N. Hunter, F. Daldal, M.C. Thurnauer, J.T. Beatty (Eds.), The Photosynthetic Purple Bacteria, Springer, The Netherladns, 2009. [4] G. McDermott, S.M. Prince, A.A. Freer, A.M. Hawthornthwaite-Lawless, M.Z. Papiz, R.J. Cogdell, N.W. Isaacs, Nature 374 (1995) 517. [5] A.W. Roszak, T.D. Howard, J. Southall, A.T. Gardiner, C.J. Law, N.W. Isaacs, R.J. Cogdell, Science 302 (2003) 1969. [6] A.P. Shreve, J.K. Trautman, H.A. Frank, T.G. Owens, A.C. Albrecht, Biochim. Biophys. Acta 1058 (1991) 280. [7] S. Hess, M. Chachisvilis, K. Timpmann, M. Jones, G. Fowler, C. Hunter, V. Sundström, Proc. Natl. Acad. Sci. USA 92 (1995) 12333. [8] H. Bergström, R. van Grondelle, V. Sundström, FEBS Lett. 250 (1989) 503. [9] K.J. Visscher, H. Bergström, V. Sundström, C.N. Hunter, R. Grondelle, Photosynth. Res. 22 (1989) 211. [10] T.J. Pflock, S. Oellerich, J. Southall, R.J. Cogdell, G.M. Ullmann, J. Köhler, J. Phys. Chem. B 115 (2011) 8813. [11] T. Pflock, M. Dezi, G. Venturoli, R.J. Cogdell, J. Köhler, S. Oellerich, Photosynth. Res. 95 (2008) 291. [12] T.J. Pflock, S. Oellerich, L. Krapf, J. Southall, R.J. Cogdell, G.M. Ullmann, J. Köhler, J. Phys. Chem. B 115 (2011) 8821. [13] S. Scheuring, R.P. Gonçalves, P. Valérie, J.N. Sturgis, J. Mol. Biol. 358 (2006) 83. [14] T. Dewa, R. Sugiura, Y. Suemori, M. Sugimoto, T. Takeuchi, A. Hiro, K. Iida, A.T. Gardiner, R.J. Cogdell, M. Nango, Langmuir 22 (2006) 5412. [15] A. Sumino, T. Dewa, M. Kondo, T. Morii, H. Hashimoto, A.T. Gardiner, R.J. Cogdell, M. Nango, Langmuir 27 (2011) 1092. [16] A. Sumino, T. Dewa, T. Takeuchi, R. Sugiura, N. Sasaki, Biomacromolecules 12 (2011) 2850. [17] A. Sumino, T. Dewa, N. Sasaki, N. Watanabe, M. Kondo, T. Morii, H. Hashimoto, M. Nango, E-J. Surf. Sci. Nanotechnol. 9 (2011) 15. [18] A.R. Varga, L.A. Staehelin, J. Bacteriol. 161 (1985) 921. [19] N.J. Russell, J.K. Coleman, T.D. Howard, E. Johnston, R.J. Cogdell, Biochim. Biophys. Acta 1556 (2002) 247.