Energy Transfer between Surface-Immobilized Light-Harvesting

Oct 21, 2010 - *Corresponding authors: Ph (+49) 6131-3924633, Fax (+49) 6131-3923787, e-mail [email protected] (H.P.); Ph (+43) 50550 4002, Fax (+4...
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Energy Transfer between Surface-Immobilized Light-Harvesting Chlorophyll a/b Complex (LHCII) Studied by Surface Plasmon Field-Enhanced Fluorescence Spectroscopy (SPFS) Rolf Lauterbach,† Jing Liu,‡, Wolfgang Knoll,*,§ and Harald Paulsen*,† Institut f€ ur Allgemeine Botanik der Johannes-Gutenberg Universit€ at, M€ ullerweg 6, 55099 Mainz, Germany, and ‡ Max-Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany. § AIT Austrian Institute of Technology, Donau City Strasse 1, 1220 Vienna. Present address: Siemens X-ray Vacuum Technology Ltd., Wuxi (SXVT ), NO. 7 Building, Land Lot 93#, Wuxi National Hi-Tech Industrial Development Zone, Wuxi 214028, Jiangsu Province, P. R. China )

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Received June 22, 2010. Revised Manuscript Received September 2, 2010 The major light-harvesting chlorophyll a/b complex (LHCII) of the photosynthetic apparatus in green plants can be viewed as a protein scaffold binding and positioning a large number of pigment molecules that combines rapid and efficient excitation energy transfer with effective protection of its pigments from photobleaching. These properties make LHCII potentially interesting as a light harvester (or a model thereof) in photoelectronic applications. Most of such applications would require the LHCII to be immobilized on a solid surface. In a previous study we showed the immobilization of recombinant LHCII on functionalized gold surfaces via a 6-histidine tag (His tag) in the protein moiety. In this work the occurrence and efficiency of F€orster energy transfer between immobilized LHCII on a functionalized surface have been analyzed by surface plasmon field-enhanced fluorescence spectroscopy (SPFS). A near-infrared dye was attached to some but not all of the LHC complexes, serving as an energy acceptor to chlorophylls. Analysis of the energy transfer from chlorophylls to this acceptor dye yielded information about the extent of intercomplex energy transfer between immobilized LHCII.

Introduction The major light-harvesting complex (LHCII) is the most abundant protein embedded in the thylakoid membrane of higher plants and green algae, binding half of their total chlorophyll (Chl) content. The knowledge of how these antenna complexes efficiently funnel absorbed sunlight energy to the reaction centers while avoiding photodamage is potentially helpful for designing artificial devices like solar cells on a molecular scale. LHCII is one of the few membrane proteins whose structure is known at high resolution.1,2 The LHCII apoprotein contains 3 R-helical transmembrane domains and noncovalently binds 14 chlorophyll and 4 carotenoid molecules. The pigment-protein complex self-organizes in vitro. When the pigments are mixed with the recombinant hydrophobic apoprotein in aqueous detergent solution, the protein spontaneously folds into a structure at least very similar to that in native LHCII.3,4 Trimerization of the complex, which can also be achieved in vitro, stabilizes LHCII toward dissociation. In its native structure, the LHCII apoprotein organizes its pigments such that the transition dipole moments of its chlorophylls are about evenly distributed over all possible directions, thus enabling it to harvest light coming in from any direction. At the same time, the pigment density is extremely high. The local Chl concentration has been estimated to *Corresponding authors: Ph (þ49) 6131-3924633, Fax (þ49) 6131-3923787, e-mail [email protected] (H.P.); Ph (þ43) 50550 4002, Fax (þ43) 50550 4000, e-mail [email protected] (W.K.). (1) Liu, Z.; Yan, H.; Wang, K.; Kuang, T.; Zhang, J.; Gul, L.; An, X.; Chang, W. Nature 2004, 428, 287–292. (2) Standfuss, R.; van Scheltinga, A. C.T.; Lamborghini, M.; K€uhlbrandt, W. EMBO J. 2005, 24, 919–928. (3) Hobe, S.; Prytulla, S.; K€uhlbrandt, W.; Paulsen, H. EMBO J. 1994, 13, 3423–3429. (4) Horn, R.; Paulsen, H. J. Mol. Biol. 2002, 318, 547–556.

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be 0.3 M,5 more than can be achieved in any organic solvent without drastically shortening excitation lifetimes due to concentration quenching.6 Moreover, the Chls in LHCII are efficiently protected toward photooxidation by rapid triplet transfer to carotenoid molecules in the complex that are also capable of relaxing singlet oxygen that has already formed.7 All these properties make LHCII interesting as a potential component in optoelectronic devices. Even if Chl possibly is intrinsically too instable for such applications, some properties of LHCII such as the self-organizing protein scaffold organizing chromophores may be worth copying into more stable artificial constructs. The best functional test for the integrity of recombinant trimeric or monomeric LHCII is the virtually complete energy transfer from Chl b to Chl a.8 Energy transfer within trimeric LHCII is rapid and leaves most of the excitation energy delocalized over a few lower energy Chl a molecules in the periphery of the complex.9,10 In the photosystem II (PSII) holocomplex, further transfer between LHCII trimers and, via the monomeric complexes CP29, CP26, or CP24, to the core complex is thought to occur in the 10-100 ps time range.11 Most of the energy transfer processes between neighboring pigment-protein complexes are lateral, in the 2D arrangement of complexes within the membrane, rather (5) Barros, T.; Kuhlbrandt, W. Biochim. Biophys. Acta, Bioenerg. 2009, 1787, 753–772. (6) Beddard, G. S.; Porter, G. Nature 1976, 260, 366–376. (7) Niyogi, K. K. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999, 50, 333–359. (8) Paulsen, H.; R€umler, U.; R€udiger, W. Planta 1990, 181, 204–211. (9) Novoderezhkin, V. I.; Palacios, M. A.; van Amerongen, H.; van Grondelle, R. J. Phys. Chem. B 2005, 109, 10493–10504. (10) Schlau-Cohen, G. S.; Calhoun, T. R.; Ginsberg, N. S.; Read, E. L.; Ballottari, M.; Bassi, R.; van Grondelle, R.; Fleming, G. R. J. Phys. Chem. B 2009, 113, 15352–15363. (11) Broess, K.; Trinkunas, G.; van Hoek, A.; Croce, R.; van Amerongen, H. Biochim. Biophys. Acta, Bioenerg. 2008, 1777, 404–409.

Published on Web 10/21/2010

DOI: 10.1021/la102525b

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than transversal between membrane layers in stacked thylakoid domains.12 In a previous study we established an artificial 2D arrangement of LHCII complexes by attaching them to a solid surface.13 In the present work we have studied the energy transfer between such surface-immobilized complexes, similar to intercomplex energy transfer in the thylakoid membrane. To this end, we attached an infrared fluorescent dye to LHCII serving as an energy acceptor for F€orster transfer from the chlorophylls. Such a construct can be viewed as a very simple biomimetic model of a photosystem, consisting of a light-harvester and an energy trap.14 We enlarged the light-harvesting system by mixing the dye-labeled LHCII complexes with nonlabeled ones on the surface. If the nonlabeled LHCII units could in fact transfer their excitation energy to the labeled ones, this was expected to be measurable as an increase in the absorption cross section of the dye in the spectral region where LHCII contributes to absorption.

Experimental Details General Materials. n-Dodecyl-β-D-maltoside (DM), EDTA, SDS, GHCl, NiCl2, sodium phosphate, biotinamidocaproate N-hydroxysuccinimide ester (NHS-biotin), and 11-mercapto-1unadecanol (spacer thiol) were purchased from Sigma-Aldrich, ethanol (HPLC grade) was from Fisher Scientific, Biotin-X-NTA was from Molecular Probes, and Hellmanex was from Hellma. Streptavidin and Biotin-thiol were kindly provided by Roche Diagnostics. NTA-terminated OEG (oligo ethylene glycol) thiol (C33H61N2O12S) and OEG spacer thiol (C22H44O5S) were kindly provided by Prof. Robert Tampe’s group (Goethe University Frankfurt).13 AlexaFluor 700 streptavidin (SA-AF700) and DY-730 were purchased from Molecular Probes and Dyomics GmbH, respectively. Labeling of LHCII with Biotin and Dye. The apoprotein of recombinant LHCII (Lhcb1) used in this work is a derivative of Lhcb1*2 (AB80)15 from pea (Pisum sativum) with Cys79 replaced by serine, with Ser160 replaced by cysteine, and with a His tag added to the C-terminus.16 Biotin was attached to the N-terminal amino group by incubating 15 μM Lhcb1 with 7.5 mM NHSbiotin in 10 mM Na phosphate buffer (pH 6.8), 0.1% (w/v) SDS for 1 h at 25 °C. The reaction was terminated by adding 2.5% (v/v) aminoethanol; the protein was precipitated by 10 mM acetic acid and adding 2.3 vol acetone, incubated for 1 h at -20 °C and centrifuged for 15 min at 14 000 rpm, 4 °C. The pellet was washed in 70% and then in 100% cold ethanol, air-dried, and stored at -20 °C. For attaching the fluorescent dye DY730 maleimide to the single cysteine, 15 μM protein in 10 mM Na phosphate (pH 7.4), 0.1% (w/v) SDS was reduced with 10 mM TCEP (tris(2-carboxyethyl)phosphine) for 20 min at 37 °C. After 1 min on ice, 10 mM DY730 in DMSO was added (15-fold molar excess over protein) and incubated for 90 min at 37 °C. The reaction was terminated by 50 mM dithiothreitol, and the protein was precipitated, washed, and stored as described above. Protein labeling efficiencies were analyzed by SDS gel electrophoresis with unlabeled proteins as control marker. Quantification of labeling efficiency was performed by using a VersaDoc Imaging System (Biorad). Reconstitution and Trimerization of LHCII. LHCII used in this study was in its trimeric form unless mentioned otherwise. Monomeric LHCII was reconstituted from its apoprotein and (12) Kirchhoff, H.; Borinski, M.; Lenhert, S.; Chi, L. F.; Buchel, C. Biochemistry 2004, 43, 14508–14516. (13) Liu, J.; Lauterbach, R.; Paulsen, H.; Knoll, W. Langmuir 2008, 24, 9661– 9667. (14) Wolf-Klein, H.; Kohl, C.; M€ullen, K.; Paulsen, H. Angew. Chem., Int. Ed. 2002, 41, 3380–3382. (15) Cashmore, A. R. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 2960–2964. (16) Horn, R.; Paulsen, H. J. Biol. Chem. 2004, 279, 44400–44406. (17) Hobe, S.; Trostmann, I.; Raunser, S.; Paulsen, H. J. Biol. Chem. 2006, 281, 25156–25166.

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pigments extracted from pea thylakoids as described earlier.17 Trimeric LHCII was made by binding reconstituted monomeric LHCII via its C-terminal His tag to a nickel-chelating fast-flow Sepharose (Amersham Biosciences) column18 and purified by sucrose gradient ultracentrifugation.19 To avoid the strong detuning effect in the surface plasmon resonance (SPR) detection of LHCII immobilization caused by high amounts of sucrose in the gradient solution of trimeric LHCII samples, the sucrose was removed by repeated ultrafiltration through a 30 kDa cutoff membrane (Microcon YM-30, Millipore), diluting the concentrated LHCII in 10 mM sodium phosphate buffer (pH 7.2), 0.05% (w/v) DM. The final LHCII concentration was assessed by using an extinction coefficient of 546 000 M-1 cm-1 at λ = 670 nm.20 Surface Preparation. Onto highly refractive LASFN9 glass slides (25252.5 mm, Schott, n=1.85 at 632.8 nm) a silver and a gold layer were evaporated. After 15 min of ultrasonic treatment in 2% Hellmanex aqueous solution, followed by 15 min of ultrasonic treatment in Milli-Q water and another 15 min in ethanol, the slides were desiccated in a stream of nitrogen and placed directly into the thermal evaporation apparatus (Edwards, FL400). The metal layers were deposited at a rate of 0.1 nm/s under ultrahigh-vacuum condition (ca. 5  106 mbar). The slides were stored separately under argon until further use within 1 week. The optimum layer thickness for both SPR and SPFS measurements was determined as 23 nm of silver and 5 nm of gold. The gold provided simultaneously a protective layer, as silver is known to be easily oxidized in air or solution. The formation of a functional layer with selective coupling capabilities on the gold surface was achieved by self-assembled monolayers (SAM) of thiolates from a binary mixture of one species with a functional end group (e.g., NTA-terminated OEG thiol) and one species with a passive hydroxyl end group (OEG spacer thiol). Glass slides were incubated with the mixture (500 μM each) dissolved in ethanol overnight at room temperature in a Teflon incubation cell. Subsequently, the slides were rinsed thoroughly with ethanol, dried in a stream of nitrogen, and then stored under an argon atmosphere. These chips were used for binding His-tagged LHCII or LHCII-DY730 under the conditions given in the figure legends. Where mentioned, an alternative surface structure was assembled upon a similar OEG thiol SAM (χbiotin-thiol =biotin thiol bound/spacer thiol bound = 0.1) by circulating a 200 nM avidin solution (Sigma), followed by a biotinylated antiavidin IgG solution (10 μg/mL, Sigma) and the avidin solution again in the flow cell for 15 min. Finally, a biotinylated antiavidin IgG solution (10 μg/mL) or a biotinylated anti-His6-tag IgG solution (10 μg/mL, Rockland Immunochemicals, Inc.) was injected and circulated for 15 min. These chips then were to bind SA-AF700 and His-tagged LHCII, respectively, as given in the figure legends. Each protein was prepared in PBST buffer, and an angular scan (see next paragraph) determining the protein binding rate was conducted at the end of a fresh PBST buffer rinse following each binding step. Instrumentation and SPFS Measurements. The laser used in this work was a Nd:YAG diode pumped solid state (DPSS) laser (CrystaLaser, 5 mW, λ = 473 nm) unless mentioned otherwise. Two modes of operation are possible: an angular scan mode in a θ-2θ reflection geometry to detect the surface-coverage dependent point of maximally enhanced electromagnetic field (resonance angle) or a kinetics mode in order to resolve timedependent processes at a fixed angle of observation. For both modes, the reflectivity channel as well as the fluorescence intensity channel can be recorded simultaneously. The setup used for SPR and SPFS measurements in this study was as described (18) Rogl, H.; Kosemund, K.; K€uhlbrandt, W.; Collinson, I. FEBS Lett. 1998, 432, 21–26. (19) Jeschke, G.; Bender, A.; Schweikardt, T.; Panek, G.; Decker, H.; Paulsen, H. J. Biol. Chem. 2005, 280, 18623–18630. (20) Butler, P. J. G.; K€uhlbrandt, W. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 2797–3801.

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Figure 1. (A) Supramolecular structure of SA-AF700 bound to biotin-LHCII on a NTA SAM surface (NTA-terminated OEG thiol/OEG spacer thiol surface). (B) Binding kinetics of LHCII on a Ni-NTA SAM surface and, as a control, on a pure OEG spacer thiol surface, detected as reflectivity changes: (X) incubation of 1 μM biotinylated LHCII in 10 mM sodium phosphate buffer (pH 7.2), 0,05% (w/v) DM, (Y) injection of a 1 mg/mL SA-AF700 stock solution (40 μg/mL final concentration), and (Z) rinse with DM-containing buffer. (C) Fluorescence emission spectrum: (1) after 60 min incubation with biotin-LHCII and (2) 10 min after SA-AF700 injection. (D) Fluorescence emission spectrum: (1) SA-AF700 bound on an avidin/antiavidin antibody/avidin/antiavidin antibody surface without LHCII for 30 min and (2) SA-AF700 bound by biotinylated LHCII on an avidin/antiavidin antibody/avidin/biotin-anti-His6 antibody surface for 10 min. previously.13 With the modified SPFS described therein, the full spectroscopic information on surface excited chromophores near the resonance angle can be obtained, which indicates the behavior of surface-bound chromophores as well as the fluorescence spectrum of homogeneously distributed chromophores in the bulk solution, which is primarily excited by the radiation of refraction and transmission below the critical angle.

Results and Discussion Energy Transfer from LHCII to an Acceptor Dye Observed by SPFS. In the photosynthetic apparatus, the light energy collected by light-harvesting complexes is conducted to the reaction centers serving as energy traps. This can be mimicked in vitro by attaching a long-wavelength fluorescent dye to LHCII as described by Wolf-Klein.14 If distances between Chls and the dye are short enough and if the donor (Chl) fluorescence emission spectrum overlaps with the acceptor (dye) absorption, then the Chls will transfer part of their excitation energy to the associated dye. In a first series of experiments we wished to test whether surface-immobilized LHCII was able to undergo excitation energy transfer to an energy acceptor that binds to the Chl-protein complexes. Surface-plasmon fluorescence spectroscopy (SPFS) was used to monitor in real time how the energy transfer was established upon addition of the acceptor dye to surface-immobilized LHCII. As the energy acceptor, the commercial near-infrared fluorescent dye AlexaFluor 700 (AF700) was used, coupled to a streptavidin molecule (SA-AF700). The streptavidin moiety enabled the acceptor dye to bind specifically to the N-terminus of the LHCII protein carrying a biotin molecule (Figure 1A). The binding kinetics of the dye-streptavidin conjugate was recorded Langmuir 2010, 26(22), 17315–17321

on an NTA SAM surface via the reflectivity channel by excitation with a HeNe laser (Uniphase, 5 mW) at 632.8 nm (Figure 1B), while the surface plasmon-excited fluorescence spectra were detected by excitation with a Nd:YAG DPSS laser at 473 nm (Figure 1C). At this wavelength, the LHCII chlorophylls are excited whereas the acceptor dye absorbs only very little. The specificity of the binding of LHCII was shown by the lack of any significant reflectivity change if the surface lacked the His tagbinding Ni-NTA group (“OEG spacer thiol surface” in Figure 1B). The fluorescence emission spectra were taken after 1 h of LHCII incubation (spectrum 1 in Figure 1C) and after 10 min upon injecting the conjugate (spectrum 2). The fluorescence change is visible after 10 min upon adding the conjugate at 730 nm (acceptor emission) as well as at 680 nm (donor emission), indicating fast binding of the streptavidin to the biotin-labeled LHCII (Figure 1C). This is consistent with the reflectivity change seen upon adding the SAAF700 conjugate, which consists of a fast and steep rise and a subsequent slower increase (Figure 1B). To exclude emission by the acceptor dye due to direct excitation by the blue laser, the emission spectrum of SA-AF700 bound to the surface (Figure 1D) was recorded without LHCII as a control (1) and compared to biotin-LHCII bound SA-AF700 (2). No AF-700 fluorescence was detected in the absence of LHCII, confirming that all emission from the fluorescent dye was sensitized by LHCII. Upon the addition of SA-AF700, the emission maximum of the donor fluorescence at 680 nm drops by about 30%. Therefore, the apparent efficiency of energy transfer between the LHCII Chls and the acceptor dye AF700, calculated as E = 1 - (donor fluorescence intensity in the presence of the acceptor/donor fluorescence DOI: 10.1021/la102525b

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intensity in the absence of the acceptor), is about 30%. The critical F€orster distance of the donor/acceptor pair Chl/AF-700, that is, the distance at which the energy transfer efficiency is expected to be 50%, is calculated as ˚ ¼ 6:1 nm R0 ¼ 9790ðΦD K2 n - 4 JDA Þ1=6 ½ A where ΦD is the fluorescence quantum yield of the donor, κ2 is the orientation factor set to 2/3, assuming free mobility of both donor and acceptor dipoles, n is the refractive index of the medium between donor and acceptor, set to 1.33, the refractive index of water, and JDA is the overlap integral of the donor emission and the acceptor absorption spectra. The distance between the N-terminus in LHCII and the nearest Chl cannot be taken from the crystal structure1,2 because N-terminal amino acids were not resolved, presumably because of their mobility. In fluorescence energy transfer measurements in detergent-solubilized LHCII this distance has been estimated to be 5.2 nm.14 The actual distance between LHCII Chls and AF700 in our setup is variable because the biotin-labeled N-terminus of the protein may bind to a biotin binding site of the streptavidin that is proximal or distal with regard to the AF700 dye attached. In any case, with an estimated donor-acceptor separation near the critical F€orster distance, the observed efficiency of energy transfer appears plausible. In Figure 1C, the amount of donor fluorescence quenching upon binding of the acceptor appears significantly smaller than the sensitized acceptor emission. This is apparent when the area between the donor emission bands at 680 nm is compared to the area between the acceptor emission bands at 710 nm. The loss of donor emission and the gain of acceptor emission should be proportional to the fluorescence quantum yields of the two chromophores which are 20%21 and 25% (information from the provider of AF-700), respectively. The under-representation of donor quenching may be explained by the shorter distance of the chlorophylls to the Au surface. If fluorophores are located at a distance of up to about 10 nm from a metal surface, some of their excitation energy will be dissipated into the metal as heat,22 and at intermediate separation distances of up to ca. 20 nm the optically excited chromophores can effectively couple back via resonance energy transfer to the plasmonic states of the metal.23 The shorter the distance of the chromophores to the surface, the more excitation energy is funneled into decay channels leading to fluorescence quenching. The distance of the LHCII chlorophylls to the metal in our surface structure (Figure 1A) is estimated to be 4-8 nm and the distance between the AF-700 dye and the surface 10-14 nm. Thus, the fluorescence quantum yield of the donor is expected to be lowered more strongly by the metal surface than that of the acceptor. This would explain why the area under the donor (Chl) emission band representing the quenched fluorescence is smaller than expected. Consistently, if the distance between metal and chromophores is increased by introducing extra layers of avidin/avidin-specific antibody/avidin/biotin-labeled anti-His6 antibody before attaching the LHCII, the Chl emission relative to the acceptor dye fluorescence is much more prominent and so is the amount of quenched donor emission (Figure 1D and Figure S1 in the Supporting Information). Intracomplex Energy Transfer to an Acceptor Dye Coupled Directly to LHCII. In the following series of experiments we tested whether a simple model of a photosynthetic photosystem (21) Gundlach, K.; Werwie, M.; Wiegand, S.; Paulsen, H. Biochim. Biophys. Acta, Bioenerg. 2009, 1787, 1499–1504. (22) Knobloch, H.; Brunner, H.; Leitner, A.; Aussenegg, F.; Knoll, W. J. Chem. Phys. 1993, 98, 10093–10095. (23) Pockrand, I.; Brillante, A.; M€obius, D. Nuovo Cimente B 1981, 63, 350.

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Figure 2. Fluorescence emission spectra at 460 nm excitation of LHCII-DY730 monomer and LHCII-DY730 trimer in sodium phosphate buffer (pH: 7.2) containing 0.1% DM.

could be immobilized on a surface, that is, an LHCII to which an efficient energy trap is covalently attached that then collects most or all of the harvested excitation energy. For this purpose, we used a different infrared fluorescent dye, DY730, that binds via its maleimide function to a singular Cys engineered in position 160 in one of the hydrophilic loop domains of LHCII. The dye’s emission with its maximum at λ=758 nm is sufficiently separated from the Chl a emission peak at λ = 680 nm to allow for its observation even at low-resolution conditions. Comparison of the protein absorbance of LHCII and LHCII-DY730 at 280 nm indicated a labeling efficiency of about 72% (data not shown). This means that 98% of all trimers carry at least one label, which, due to the close coupling of Chls within a trimer,10 then serves as an energy trap for the entire trimeric complex. Consistently, in detergent-solubilized labeled LHCII, the donor (Chl) fluorescence at 680 nm is quenched in the presence of the acceptor dye by 65 and 95% in monomers and trimers, respectively (Figure 2). The corresponding stronger increase in acceptor dye emission, maximum around 760 nm, in trimers compared to monomers confirms that the changes in fluorescence intensities in fact are due to F€orster energy transfer. Intercomplex Energy Transfer Observed by SPFS. If LHCII is bound on the surface at a sufficient density, then the complexes should be able to exchange excitation energy. To verify this, we mixed the energy trap-labeled LHCII described in the last section with nonlabeled LHCII. In the presence of intercomplex energy transfer, the acceptor dyes representing the energy traps should be able to collect excitation energy from several neighboring, nonlabeled light-harvesting complexes, similar to the reaction centers in the thylakoid membrane. The spectra of surface-immobilized LHCII and surfaceimmobilized LHCII-DY730 (spectra 1 and 2, respectively, in Figure 3) on the surface (panel A) are very similar to the ones measured in solution (panel B and Figure 2), demonstrating that the intracomplex energy transfer from Chls to DY730 is about the same in both situations. This is not true for the 3:7 mixture of labeled and nonlabeled complexes (spectrum 3). In solution (panel B), the spectrum measured (3) perfectly coincides with the theoretical spectrum (4), composed of 0.7 spectrum (1) plus 0.3 spectrum (2). If the mixture is bound to the surface (panel A), then the sensitized acceptor emission measured at around 760 nm exceeds the intensity expected from the theoretical contribution of the two complexes, and the donor emission at around 680 nm is somewhat further quenched compared to the theoretical value. This indicates additional energy transfer from Chls to DY730, exceeding the intramolecular transfer in LHCII-DY730. Langmuir 2010, 26(22), 17315–17321

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Figure 3. Fluorescence emission spectra at 473 nm excitation of (A) surface-bound LHCII and (B) bulk LHCII in solution. Spectrum (1), 1 μM LHCII; spectrum (2), 1 μM LHCII-DY730; spectrum (3), mixture of 30% LHCII-DY730 and 70% LHCII, total concentration 1 μM; spectrum (4), theoretical spectrum in the absence of intercomplex energy transfer, composed of 0.7 spectrum (1) and 0.3 spectrum (2); spectrum (5), background; In (A), spectra were taken after incubating the LHCII solutions for 30 min with a freshly regenerated chip surface. The spectra in panel (B) are all taken at an incident angle of θ = 45° at which no surface-plasmon field enhancement occurs and a small part of the light is transmitted into the water medium, exciting the fluorophores in the bulk solution and at the metal-aqueous interface simultaneously. Since the complexes coupled to the surface account for only a small fraction of the total protein in the bulk phase, the fluorescence spectra in panel (B) reflect the situation in solution.

We conclude that on the surface but not in solution nonlabeled LHCII complexes are able to transfer part of their excitation energy to an acceptor dye attached to a neighboring LHCIIDY730. No intercomplex energy transfer is expected to take place in detergent solution since the mean distance between LHCII molecules is too large for F€orster energy transfer to take place, whereas on the surface the complexes are expected to be bound at shorter distances. Quantitation of Intercomplex Energy Transfer. We tested the notion of intercomplex energy transfer between surfacebound LHCII by using different mixtures of the labeled and nonlabeled complex. The smaller the fraction of labeled LHCII, the more unlabeled complexes will be in the vicinity of each acceptor dye that do not have an energy trap of their own and, therefore, can transfer their excitation energy to the nearest acceptor dye. Thus, with smaller fractions of labeled material on the surface, the additional sensitized acceptor dye fluorescence and quenching of donor (Chl) fluorescence, both due to intercomplex energy transfer, should become more pronounced. A prerequisite to conduct comparable intercomplex energy transfer measurements is that the surface chemistry used for immobilizing the complexes is reliable enough to capture the same amounts of protein in each subsequent regeneration-binding cycle. As shown in our previous study, this is the case with only an estimated 20% deviation over four cycles.13 Even so, as a matter of caution, the same chip was never reused more often than twice. For a quantitative assessment of intercomplex energy transfer, we compared the ratio of donor quenching as well as sensitized acceptor fluorescence between surface and solution at various ratios of LHCII-DY730 and LHCII as in Figure 3. To do this on a quantitative level, the fluorescence emission spectra of LHCIIDY730 and LHCII mixtures on the chip and in solution needed to be deconvoluted, using spectra of LHCII (containing only the donor chromophore chlorophyll) and of Lhcb1-DY730 (containing only the acceptor dye) as templates. Figure 4 shows an example of Langmuir 2010, 26(22), 17315–17321

Figure 4. Deconvolution of the fluorescence emission spectrum of an LHCII-DY730/LHCII mixture (1) into its components LHCII (donor) emission (2) and Lhcb1-DY730 (acceptor) emission (3). Spectra (2) and (3) were fitted such that their sum (4) approximates spectrum (1).

such a deconvolution. The sum (4) of the fitted donor (2) and acceptor (3) emissions matches the spectrum of the LHCII-DY730/ LHCII mixture (1) very well, indicating a reasonable deconvolution fit. The integrated areas under spectra 2 and 3 were taken as a measure of donor and acceptor fluorescence emission, respectively. The spectra of the pure components were measured on the same chip as each mixture. The donor quenching was then calculated as the ratio of the donor emission of the mix divided by the pure LHCII emission on surface and in solution. The sensitized acceptor fluorescence was calculated as the ratio of acceptor emissions of the mix divided by the pure LHCII-DY730 emission on surface and in solution. DOI: 10.1021/la102525b

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Figure 5. Increase of (A) donor quenching and (B) sensitized acceptor emission due to intercomplex energy transfer between surfaceimmobilized LHCII complexes. Each data point represents the measurement of surface-bound LHCII divided by the measurement in solution, both at the given mixture of LHCII and LHCII-DY730. Data points with error bars are based on three independent measurements each.

The energy transfer efficiency corresponds to the extent of donor emission quenching in the presence of the acceptor. For pure dye-labeled LHCII in solution or immobilized on the chip surface, then donor quenching amounts to 95%; this is the intramolecular energy transfer efficiency Qi. If only a fraction of χ LHCII molecules is labeled and if we assume that there is no intermolecular energy transfer, then the theoretical donor quenching would be χQi. This is the amount of donor quenching observed in solution where the complexes do not undergo intercomplex energy transfer (Figure 3). If in surface measurements the actual donor quenching observed at a given fraction of labeled LHCII, Qo, is larger than χQi, then this indicates intercomplex energy transfer. The rise factor of donor quenching (RFQ), due to energy transfer from nonlabeled to labeled LHCII, would then be RFQ = Qo/(χQi). In Figure 5A, RFQ is shown for various ratios χ of labeled and nonlabeled LHCII. As expected, RFQ increases at lower χ since the acceptor dye can collect more energy from surrounding LHCII complexes if fewer of them carry acceptor dyes of their own. If every tenth LHCII carries a label, then RFQ is 2.3 ( 0.8; i.e., every acceptor dye collects energy from roughly two light-harvesting complexes. The observation of Qo > χQi cannot be explained by the quenching effect of the Au surface since this is constant and independent of the presence or absence of the acceptor dye. A similar algorithm can be used for the increase of sensitized acceptor fluorescence due to intermolecular energy transfer between LHCII complexes. If Ei is the sensitized acceptor emission observed with pure dye-labeled LHCII and Eo the sensitized acceptor emission observed at a given fraction of LHCII-DY730, then the rise factor of sensitized acceptor emission due to intercomplex energy transfer, RFE = Eo/(Eiχ). As seen in Figure 5B, the rise factor of sensitized acceptor fluorescence also increases, similar to the rise factor of donor quenching, if less and less LHCII carry an acceptor dye, reaching a value of about 3 at χ = 0.1. RFE seems slightly higher than RFQ at higher dilutions of the labeled complex (compare parts A and B of Figure 5 at χ e 0.3) although this difference is not significant. The fact that RFE does not approach 1 at higher χ values (Figure 5B at χ g 0.5) is most likely due to an error in subtracting the LHCII fluorescence contribution, which is relatively large as compared to the dye emission under these conditions (see Figure 3A). Possible Limitations of Intercomplex Energy Transfer between Surface-Immobilized LHCII. Because of the architecture 17320 DOI: 10.1021/la102525b

of photosystem II in the photosynthetic apparatus of green plants, most excitons will have to pass several light-harvesting complexes before they reach the energy trap, the reaction center.24,11 The quantum yield of energy transfer to the reaction center is not far from 100% under favorable physiological conditions; therefore, intercomplex energy transfer must be highly efficient in the native photosynthetic membrane. In fact, surface-immobilized lightharvesting complexes LH2 from the photosynthetic apparatus of the purple bacteria Rhodobacter sphaeroides have recently been shown to transfer excitation energy efficiently over distances in the micrometer range,25 much more than what is required in photosynthesis. The ring-shaped complexes, each comprising 27 bacteriochlorophyll (BChl) molecules, were immobilized on a glass surface either as 2D crystals or in nearly linear arrays of several micrometers length.26 Upon excitation of only a small section of immobilized LH2, BChl fluorescence emission was detected up to 2 μm away from the excitation area. If the surfaceimmobilized LHCII was equally efficient in transferring its excitation energy, then each complex should be able to transfer its energy to the acceptor dye serving as the energy trap. This clearly is not the case. If every tenth LHCII carries an acceptor dye, then the absorption cross section of the dye, compared to that of a dye attached to an isolated LHCII, does not rise by a factor of 10 but only of 2-3. Two principal reasons may be responsible for this observation. (1) Energy transfer between complexes occurs with low efficiency because they are too far apart from another or because they are positioned such that the low-energy Chl a molecules are too far apart. (2) There are other energy traps in the system that compete with the acceptor dyes. To determine the distances between surface-coupled LHCII in our setup, the molecular mass and size of the complex need to be known. This includes the belt of detergent molecules shielding the hydrophobic, transmembrane section of each LHCII. With an estimated 50 kDa contribution by the DM micelles, the total mass per LHCII complex is about 170 kDa. The footprint of trimeric LHCII can be approximated as a circular area with a diameter of (24) Boekema, E. J.; van Roon, H.; van Breemen, J. F.L.; Dekker, J. P. Eur. J. Biochem. 1999, 266, 444–452. (25) Escalante, M.; Lenferink, A.; Zhao, Y.; Tas, N.; Huskens, J.; Hunter, C. N.; Subramaniam, V.; Otto, C. Nano Lett. 2010, 10, 1450–1457. (26) Escalante, M.; Zhao, Y. P.; Ludden, M. J.; Vermeij, R.; Olsen, J. D.; Berenschot, E.; Hunter, C. N.; Huskens, J.; Subramaniam, V.; Otto, C. J. Am. Chem. Soc. 2008, 130, 8892–8893.

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6.3 nm as taken from the crystal structure.1 As DM micelles have been described as oblate ellipsoids with a radius ranging from ca. 1.7 to 3.4 nm,27,28 the complex-associated detergent belts are estimated to add about 4 nm to the diameter of the complex. The diameter of the LHCII complexes including their detergent belt is therefore estimated to be about 10.3 nm. The protein mass density with 100% coverage of an LHCII monolayer on the surface is then calculated as 1 mm2  170 kg=mol ¼ 3:4 ng=mm2 π  5:15 nm2  6:02  1023 mol - 1 This equals a 0.65° SPR minimum angle shift in our setup.29 LHCII coupling on the NTA SAM (χ=0.4) surface produced an angular shift of up to about 0.5°, corresponding to roughly 75% surface coverage. This calculation assumes binding of LHCII to the surface in a monolayer which is justified by the strict dependence of LHCII binding on the interaction between a His tag in the protein and a Ni-NTA group on the surface (Figure 1B). If the footprints of detergent-solubilized LHCII complexes are simplified to circles, then their densest (hexagonal) packing on a planar surface would cover almost 91% of the available area. An overall separation between bound complexes of 1 nm would reduce their surface coverage by nearly 20%, which approximates the observed overall coverage of 75%. The shortest possible Chl-Chl distance between complexes would then be 5 nm. Taking into account that the Chls involved in intercomplex energy transfer need not necessarily be the ones closest to the neighboring complexes, the distance would even be larger. This may in fact be limiting for efficient energy transfer between Chl a molecules, since the donor/acceptor pair LHCII/Chl a exhibits a critical F€orster distance of ∼5 nm. The linear LH2 arrays, too, were made in an aqueous solution containing DM as a detergent;25 however, the host-guest interaction used to mediate LH2 immobilization may have helped to keep the distances between LH2 rings short enough for highly efficient energy transfer. The other possibility to explain limited complex-complex energy transfer on the surface, the presence of energy traps other than the acceptor dye, is also worth considering. In plants, LHCII is involved in a process called nonphotochemical quenching (npq) in which excess excitation energy is dissipated as heat.30 This process in vivo is induced by a low pH value on the lumenal side of the thylakoid membrane and a number of components, neither of which is present in or around our immobilized LHCII; therefore, npq is unlikely to play a role in our system. Isolated LHCII has been described to be able autonomously to adopt a quenching conformation,31 but this has been questioned by others.32 However, even pure preparations of LHCII contain traces of unbound Chls. These tend to aggregate with one another or with peripheral Chls in LHCII, and such Chl aggregates dissipate excitation (27) Dupuy, C.; Auvray, X.; Petipas, C. Langmuir 1997, 13, 3965–3967. (28) Timmins, P. A.; Leonhard, M.; Weltzien, H. U.; Wacker, T.; Welte, W. FEBS Lett. 1988, 238, 361–368. (29) Yu, F. Surface Plasmon Fluorescence Spectroscopy and Surface Plasmon Diffraction in Biomolecular Interaction Studies. Thesis/Dissertation, Max-PlanckInstitut f€ur Polymerforschung, 2004. (30) M€uller, P.; Li, X.; Niyogi, K. Plant Physiol. 2001, 125, 1558–1566. (31) Pascal, A. A.; Liu, Z. F.; Broess, K.; van Oort, B.; van Amerongen, H.; Wang, C.; Horton, P.; Robert, B.; Chang, W. R.; Ruban, A. Nature 2005, 436, 134– 137. (32) Barros, T.; Royant, A.; Standfuss, J.; Dreuw, A.; K€uhlbrandt, W. EMBO J. 2009, 28, 298–306.

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Article

energy very rapidly.6 This effect has been discussed to explain the apparently “quenched state” of LHCII crystals. Among the closely coupled LHCII trimers in the crystal, the Chl aggregates can dissipate the excitation energy of a large number of complexes.32 Similarly, such Chl aggregates present in the 2D arrangement of LHCII studied in this work may compete with the acceptor dyes as energy traps. In measurements of purely labeled LHCII attached to the surface, the dissipating aggregates would not be noticeable since the quenched complexes are not visible in the fluorescence emission and therefore do not affect the intramolecular energy transfer observed. Only if the labeled LHCII is mixed with nonlabeled complexes, then the Chl aggregates would dissipate the excitation energy of some of the nonlabeled complexes and, thus, keep them from transferring their energy to the acceptor dyes nearby. Both of these potential limitations of efficient complexcomplex energy transfer may be overcome by embedding the surface-bound LHCII complexes in a biological membrane. This membrane may be immobilized on the surface by using surfacebound LHCII as tethers as has been shown with the membrane protein cytochrome c oxidase.33,34 In the hydrophobic space of the membrane, the individual LHCII complexes would be able to make direct hydrogen-bond contacts, similar to the chlorophyll containing complexes in the thylakoid membrane, and, thus, get closer to one another and exchange energy more efficiently. Moreover, Chl molecules eventually dissociating from LHCII complexes would still be dissolved in the hydrophobic phase of the membrane and therefore be less prone to undergo aggregation than in the aqueous detergent environment.

Conclusions In this work we have demonstrated that surface-immobilized LHCII is able to undergo intercomplex energy transfer. This is a prerequisite for using these or similar pigment-protein complexes in devices such as photovoltaic applications as efficient light harvesters or for using arrays of surface-bound LHCII for conducting excitation energy. Such arrays of the bacterial lightharvesting complex LH2 have been shown to conduct excitation energy over distances in the micrometer range.25 The energy transfer efficiency between immobilized LHCII complexes is clearly detectable but relatively low and far from the near-unity transfer efficiency in the thylakoid membrane where plant photosynthesis takes place. This efficiency may be approximated more closely if the immobilized LHCII complexes are embedded in a tethered membrane rather than in detergent micelles. Experiments along these lines are underway. Acknowledgment. This work was funded in part by BMBF (Centre for Multifunctional Materials and Miniaturized Devices, TA1, to H.P.). Supporting Information Available: Figure S1. This material is available free of charge via the Internet at http://pubs. acs.org. (33) Giess, F.; Friedrich, M. G.; Heberle, J.; Naumann, R. L.; Knoll, W. Biophys. J. 2004, 87, 3213–3220. (34) Ataka, K.; Giess, F.; Knoll, W.; Naumann, R.; Haber-Pohlmeier, S.; Richter, B.; Heberle, J. J. Am. Chem. Soc. 2004, 126, 16199–16206.

DOI: 10.1021/la102525b

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