Supramolecular Organization of the Main Photosynthetic Antenna

Apr 21, 2009 - §Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center,. Fort Worth, Texas, and...
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Supramolecular Organization of the Main Photosynthetic Antenna Complex LHCII: A Monomolecular Layer Study Wiesleaw I. Gruszecki,*,† Ewa Janik,‡ Rafal Luchowski,†,§ Peter Kernen, Wojciech Grudzinski,† Ignacy Gryczynski,§ and Zygmunt Gryczynski§ †

Department of Biophysics, Institute of Physics, Maria Curie-Skleodowska University, 20-031 Lublin, Poland, Department of Plant Physiology, Institute of Biology, Maria Curie-Skleodowska University, Lublin, Poland, § Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center, Fort Worth, Texas, and Zyomyx Inc., Hayward, California )

‡

Received February 20, 2009. Revised Manuscript Received March 27, 2009 The light-harvesting pigment-protein complex LHCII is a main antenna complex of the photosynthetic apparatus of plants, responsible for collecting light energy and also for photoprotection against overexcitation-induced damage. Realization of both functions depends on molecular organization of the complex. Monolayer technique has been applied to address the problem of supramolecular organization of LHCII. Analysis of the isotherms of compression of monomolecular films formed at the argon-water interface shows that LHCII appears in two phases: one characterized by the specific molecular area characteristic of trimeric and one of monomeric organization of LHCII. Monolayers of LHCII were deposited by means of the Langmuir-Blodgett technique to solid supports and examined by means of AFM, FTIR, fluorescence spectroscopy, and fluorescence lifetime imaging microscopy (FLIM). FTIR analysis shows that organization of the trimers of LHCII within a monolayer is associated with formation of intermolecular hydrogen bonds between neighboring polypeptides. The linear-dichroism FTIR analysis reveals that polypeptide fragments involved in intermolecular interactions are oriented at an angle of 67 with respect to the normal axis to the plane of the layer. Fluorescence and fluorescence lifetime analysis reveal that the organization of LHCII within monolayers is associated with formation of the low-lying excitonic energy levels that can be potentially responsible for excess excitation quenching. FLIM and AFM reveal heterogeneous organization of LHCII monolayers, in particular, formation of ringlike structures. The potential of LHCII to form molecular structures characterized by pigment excitonic interactions is discussed in terms of regulation of the photosynthetic accessory function and photoprotection against overexcitationinduced damage.

1. Introduction The major photosynthetic light-harvesting pigment-protein complex LHCII is the most abundant membrane protein in thylakoid membranes, comprising more than half of the pool of chlorophyll pigments in the biosphere.1,2 In vivo, LHCII appears as a trimer composed of three Lhcb proteins (Lhcb1-3).1,2 Each monomer comprises 8 molecules of chlorophyll a, 6 molecules of chlorophyll b, and 4 molecules of xanthophyll pigments: 2 luteins, 1 neoxanthin, and 1 violaxanthin.2 Apart of the main physiological function of LHCII, namely, absorption of light quanta and transferring electronic excitation energy to the photosynthetic reaction centers, the complex plays an important structural role in maintaining the functional state of the thylakoid membrane and the thylakoid membrane system.3 Another biological function of LHCII, considered to be a crucial one, is associated with maintaining the integrity of the photosynthetic apparatus, under conditions of light stress leading to over-excitation of the reaction centers and the entire antenna system. Under such conditions, a molecular mechanism of feedback de-excitation is activated, resulting in massive thermal energy dissipation.4 There is no *Corresponding author. E-mail: [email protected]. Fax: + 4881 537 61 91. :: (1) Kuhlbrandt, W.; Wang, D. N.; Fujiyoshi, Y. Nature (London) 1994, 367, 614. (2) Liu, Z.; Yan, H.; Wang, K.; Kuang, T.; Zhang, J.; Gui, L.; An, X.; Chang, W. Nature (London) 2004, 428, 287. (3) Standfuss, R.; van Scheltinga, A. C. T.; Lamborghini, M.; Kuhlbrandt, W. EMBO J. 2005, 24, 919. (4) Cogdell, R. J. Trends Plant Sci. 2006, 11, 59.

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doubt about a vital role of LHCII in this process, although there are several concepts regarding detail mechanisms operating at the molecular level, involved in switching between the photosynthetic antenna function and photoprotection. Among the popular concepts forwarded recently, there is light-induced structural transformation of the antenna protein, associated with change in configuration of the bound neoxanthin, leading to efficient excitation quenching by another protein-bound xanthophyll lutein.5 Another mechanism proposed is directly based on formation of the chlorophyll-xanthophyll (zeaxanthin) charge transfer heterodimer, leading to efficient excessive excitation quenching.6 In addition to the role of the LHCII-bound xanthophylls in direct chlorophyll triplet excitation quenching,7 a direct role of zeaxanthin in physical quenching of chlorophyll singlet excitations has also been postulated recently by Standfuss and coworkers, based on the analysis of the X-ray crystallographydetermined structure of LHCII.3 Chlorophyll excitation quenching can even be observed in a sample composed of trimeric LHCII,8 but association of LHCII trimers into more organized structures results in pronounced quenching of singlet excitations (5) Ruban, A. V.; Berera, R.; Ilioaia, C.; van Stokkum, I. H.; Kennis, J. T.; Pascal, A. A.; van Amerongen, H.; Robert, B.; Horton, P.; van Grondelle, R. Nature (London) 2007, 450, 575. (6) Holt, N. E.; Zigmantas, D.; Valkunas, L.; Li, X. P.; Niyogi, K. K.; Fleming, G. R. Science 2005, 307, 433. (7) Mozzo, M.; Dall’Osto, L.; Hienerwadel, R.; Bassi, R.; Croce, R. J. Biol. Chem. 2008, 283, 6184. (8) Ilioaia, C.; Johnson, M. P.; Horton, P.; Ruban, A. V. J. Biol. Chem. 2008, 283, 29505.

Published on Web 04/21/2009

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and Fourier transformed. Absorption spectra in the region between 4000 and 600 cm-1, at a resolution of one data point every 2 cm-1, were obtained using a clean crystal as the background. ATR crystals were cleaned with organic solvents. Wire grid infrared polarizer KRS-5 (Pike Technologies, USA) was used in the IR linear dichroism experiments. Spectral analysis was performed with OPUS software (Bruker, Germany) and Grams 32 software from Galactic Industries (USA). FTIR technique with polarization of the IR beam was used to obtain information on molecular order and orientation.24,25 This is based on the fact that the absorbance (A) is proportional to the square of the product of the transition dipole moment ( μ) and the projection of the electric field (E) of the polarized IR beam on the direction of the transition dipole:

2. Materials and Methods 2.1. LHCI Isolation. The largest light-harvesting antenna

2.2. Monomolacular Layer Preparation and Film Deposition. Monomolecular layers of LHCII were prepared in a Teflon trough at the argon-water interface according to the procedure described in detail previously.16,18,23 The Tricine buffer, as used for LHCII suspension, was also used as a subphase. The specific resistivity of water used to prepare buffer for the subphase was typically higher than 15 MΩ cm. Monolayers of LHCII were deposited to solid supports at constant computercontrolled surface pressure by means of the Langmuir-Blodgett technique according to the protocol described in detail previously.16,18,23 Monolayers have been deposited to either ZnSe monocrystal trapezoid, for ATR-FTIR measurements, to freshly cleaved mica for AFM scanning and to nonfluorescence glass slides or mica slides for fluorescence measurements. Monolayer compression and deposition was carried out at 25 ( 1 C. 2.3. FTIR Measurements. Infrared absorption spectra were recorded with the Fourier transform infrared (FTIR) spectrometer, model Vector 33 from Bruker (Germany). Before measurements (40 min) and during all measurements, the instrument was purged with dry argon. The attenuated total reflection (ATR) configuration was used with a ten-reflection ZnSe crystal (45 cut). Typically, ten interferograms were collected, averaged, (9) Gruszecki, W. I.; Grudzinski, W.; Gospodarek, M.; Patyra, M.; Maksymiec, W. Biochim. Biophys. Acta 2006, 1757, 1504. (10) Horton, P.; Wentworth, M.; Ruban, A. FEBS Lett. 2005, 579, 4201. (11) Yan, H.; Zhang, P.; Wang, C.; Liu, Z.; Chang, W. Biochem. Biophys. Res. Commun. 2007, 355, 457. (12) Lambrev, P. H.; Varkonyi, Z.; Krumova, S.; Kovacs, L.; Miloslavina, Y.; Holzwarth, A. R.; Garab, G. Biochim. Biophys. Acta 2007, 1767, 847. (13) Shao, L.; Konka, V. V.; Leblanc, R. M. J. Colloid Interface Sci. 1999, 215, 92. (14) Gallant, J.; Lavoie, H.; Tessier, A.; Munger, G.; Leblanc, R. M.; Salesse, C. Langmuir 1998, 14, 3954. (15) Grudzinski, W.; Matula, M.; Sielewiesiuk, J.; Kernen, P.; Krupa, Z.; Gruszecki, W. I. Biochim. Biophys. Acta 2001, 1503, 291. (16) Gruszecki, W. I.; Grudzinski, W.; Banaszek-Glos, A.; Matula, M.; Kernen, P.; Krupa, Z.; Sielewiesiuk, J. Biochim. Biophys. Acta 1999, 1412, 173. (17) Gruszecki, W. I.; Grudzinski, W.; Matula, M.; Kernen, P.; Krupa, Z. Photosynth. Res. 1999, 59, 175. (18) Kernen, P.; Gruszecki, W. I.; Matula, M.; Wagner, P.; Ziegler, U.; Krupa, Z. Biochim. Biophys. Acta 1998, 1373, 289. (19) Liu, J.; Lauterbach, R.; Paulsen, H.; Knoll, W. Langmuir 2008, 24, 9661. (20) Krupa, Z.; Huner, N.; Williams, J.; Maissan, E.; James, D. Plant Physiol. 1987, 84, 19. (21) Gruszecki, W. I.; Gospodarek, M.; Grudzinski, W.; Mazur, R.; Gieczewska, K.; Garstka, M. J. Phys. Chem. B 2009, 113, 2506. (22) Grudzinski, W.; Krupa, Z.; Garstka, M.; Maksymiec, W.; Swartz, T. E.; Gruszecki, W. I. Biochim. Biophys. Acta 2002, 1554, 108. (23) Gruszecki, W. I. Methods Mol. Biol. 2004, 274, 173.

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ð1Þ

)

where j is the angle between the directions of μ and E. The dichroic ratio (R) is defined as a ratio of the integrated absorption bands or the absorbance values corresponding to the selected vibrational modes in the parallel, A , and perpendicular, A^, configurations: R A ðνÞ dν A cos2 j ¼ ¼R A^ A^ ðνÞ dν cos2 j^

)

R ¼

)

complex, LHCII, was isolated from fresh spinach leaves according to procedure of Krupa and co-workers,20 which was slightly modified.21 Purity of the preparation has been tested by means of electrophoresis, mass spectrometry, and pigment chromatography.21 The preparation was suspended in a Tricine buffer (20 mM, pH 7.6) containing 10 mM KCl. The preparation presented typical LHCII electronic absorption, fluorescence, and FTIR spectra.9,17,21-23

Α ¼ jμEj2 cos2 j

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and has been proposed to be one of the important mechanisms of photoprotection in the photosynthetic apparatus realized at the molecular level.9-11 The importance of the intertrimer interactions in the LHCII assemblies in vivo has been concluded on the basis of the circular dichroism and time-resolved fluorescence comparative analyses of the protein in model systems and in thylakoid membranes.12 In the present study, we apply the monomolecular layer model system to study formation of supramolecular structures by LHCII trimers and to analyze intermolecular interactions. Monomolecular layer system have been successfully applied in studies of photosynthetic proteins,13,14 including LHCII.15-19

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The procedure applied in the present work is described in detail in our previous work.26-28 2.4. AFM Measurements. Scanning force microscopy (called atomic force microscopy, AFM) was carried out in tapping mode on a BioScope (Digital Instruments, Santa Barbara, CA) with scan speeds around 1 Hz. Silicon tips with resonance frequencies between 315 and 375 kHz (Digital Instruments TESP, L = 12.5 μm) have been used for imaging. All AFM images are raw data except corrected for image leveling. More details of scanning are described elsewhere.18

2.5. Fluorescence, Fluorescence Lifetime, and FLIM Measurements. Fluorescence spectra were recorded on Cary Eclipse (Varian Inc., Australia) fluorometer. Spectra were corrected for lamp intensity and photomultiplier sensitivity. 77 K fluorescence spectra were recorded with the system described previously.15 In the case of the fluorescence excitation spectra, the excitation and emission slits were set to 5 and 10 nm, respectively, and in the case of fluorescence emission spectra, the excitation and emission slits were set to 10 and 5 nm, respectively. Fluorescence lifetimes were measured using time-correlated single photon counting (TCSPC) system FluoroTime 200 (PicoQuant, GmbH, Germany) equipped with a multichannel plate detector (MCP-PMT from Hamamatsu, Japan); a 470 nm laser diode was used for excitation, and the emission was collected through a monochromator. The TimeHarp 300 in a TCSPC system from Picoquant uses 65 536 channels with the highest resolution of 4 ps per channel. The pulse width of the used excitation laser diode is about 60 ps and time response of MCPPMT is about 30 ps (as estimated with 100 fs excitation pulse from Ti:sapphire laser system). The instrument response function is 60 ps for the excitation wavelength used in the experiments. With this instrument response function and 4 ps temporal resolution, signal deconvolution analysis allows (24) Goormaghtigh, E.; Raussens, V.; Ruysschaert, J. M. Biochim. Biophys. Acta 1999, 1422, 105. (25) Tamm, L. K.; Tatulian, S. A. Q. Rev. Biophys. 1997, 30, 365. (26) Gagos, M.; Gabrielska, J.; Dalla Serra, M.; Gruszecki, W. I. Mol. Membr. Biol. 2005, 22, 433. (27) Herec, M.; Gagos, M.; Kulma, M.; Kwiatkowska, K.; Sobota, A.; Gruszecki, W. I. Biochim. Biophys. Acta 2008, 1778, 872. (28) Sujak, A.; Gagos, M.; Serra, M. D.; Gruszecki, W. I. Mol. Membr. Biol. 2007, 24, 431.

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statistically significant lifetime determination below 10 ps. Fluorescence lifetime measurements were done with a magic angle conditions. Fluorescence lifetime imaging microscope (FLIM) images were collected with the MT 200 system from PicoQuant GmbH (Berlin, Germany) coupled with Olympus IX71 microscope and PerkinElmer APD (SPCM-AQR-14) detection system. Excitation was with 470 nm laser diode and emission was detected after the broad band-pass filter 600-800 nm. The laser repetition rate was 20 MHz and time resolution was better that 5 ps. Pure mica and glass supports were scanned as a control.

3. Results and Discussion Figure 1 presents the surface pressure-mean molecular area dependency of compression of the monomolecular layer formed with LHCII at the argon-water interface. The straight line fitted to the linear portion of the isotherm, corresponding to the surface pressure region above 35 mN/m, extrapolated to zero surface pressure, indicates the specific molecular area of 1395 A˚2. This specific molecular area is close to a value obtained previously in the case of the complex isolated from winter rye (1433 A˚2).18 Taking into account the fact that the LHCII molecule has an elliptical cross section2,29 characterized by the minor and major axes of 30 A˚ and 50 A˚, one can expect a specific molecular area within the range 1178-1500 A˚2, in the case of monomeric organization of the complex in the monolayer. Molecular organization patterns considered to be “tight” shell yield in molecular areas close to the area of the elliptical cross section (1178 A˚2). Trimeric organization of the LHCII shell yields specific molecular areas within the range 2618-3333 A˚2. The low limit corresponds to the mean area of the monomer within the cylinder-shaped trimer, and the high limit value corresponds to the mean area of the monomer in the 100  100 A˚ rectangle. Interestingly, the shape of the isotherm reveals that upon increasing the surface pressure one is able to induce a transition from the trimeric to monomeric organization of LHCII. On the other hand, the transition observed is not very sharp, which means that both the molecular organization forms can be present both at higher and lower surface pressure values. The physiological importance of such a transition in the LHCII organization (trimer-monomer transition) has been discussed as potentially induced by temperature rise30 or by illumination with strong light.31 Owing to the fact that the transition is observed at ca. 33 mN/m (see Figure 1), and therefore in the region typical of natural biomembrane lateral pressure values (∼30-35 mN/m),32 it is possible that the monomer-trimer transition can be induced by local lateral pressure changes related, for example, to inhomogeneous lipid composition or protein crowding. Interestingly, the monomolecular layer study has revealed that at both the surface pressure values typical of trimeric and monomeric LHCII the protein undergoes thermal conformational transition, in the temperature range between 21 and 25 C.17 Figure 2 presents the IR absorption spectrum, in the amide I region, of the LHCII preparation and superimposed spectra of the LHCII monomolecular films deposited at the surface pressures characteristic of the trimeric (15 mN/m) and monomeric (35 mN/m) organizations of the complex. The band originates mostly (at ca. 90%) from the molecular vibrations of the protein :: (29) Kuhlbrandt, W.; Wang, D. N. Nature (London) 1991, 350, 130. (30) Yang, C.; Boggasch, S.; Haase, W.; Paulsen, H. Biochim. Biophys. Acta 2006, 1757, 1642. (31) Garab, G.; Cseh, Z.; Kovacs, L.; Rajagopal, S.; Varkonyi, Z.; Wentworth, M.; Mustardy, L.; Der, A.; Ruban, A. V.; Papp, E.; Holzenburg, A.; Horton, P. Biochemistry 2002, 41, 15121. (32) Marsh, D. Biochim. Biophys. Acta 1996, 1286, 183.

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Figure 1. Surface pressure-mean molecular area isotherm of compression of the monomolecular layer formed with LHCII at the argon-water interface. The straight dashed lines fitted to the linear portions of the isotherm, extrapolated to the zero surface pressure, indicate the specific molecular areas characteristic of monomeric (1395 A˚2) and trimeric (3060 A˚2) molecular organization. See the text for more explanation.

Figure 2. Infrared absorption spectra in the amide I region of LHCII preparation used to prepare monolayers (solid line) and of LHCII in monomolecular layers deposited by means of the Langmuir-Blodgett technique to ZnSe monocrystal, at the surface pressure 15 and 35 mN/m, indicated. Panel A, original spectra normalized at the absorption maximum; panel B, difference spectra monolayer minus bulk protein (as indicated).

component of LHCII. As can be seen from the analysis of the amide I band, the formation of LHCII monolayers preserves the native structure of the protein. This can be concluded on the basis of relatively small spectral differences between the original LHCII Langmuir 2009, 25(16), 9384–9391

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used to deposit a film and the monolayer. The maximum of the amide I band (at ca. 1650 cm-1) corresponds to the principal R-helical components of the protein.9,25 In addition, contribution from the bands assigned to turns and loops (1670-1680 cm-1) antiparallel (at ca. 1684 and 1695 cm-1) and parallel (at ca. 1620 cm-1) β-structures and the low-energy band attributed to protein aggregates strands (at ca. 1610 cm-1)9,25 can be observed (see also Figure 3). The minor component assigned to antiparallel strands corresponds most probably to such a structure identified in the luminal loop.33 The presence of the bands that can be formally attributed to parallel β-strands is associated with formation of molecular structures of proteins, stabilized by intermolecular hydrogen bonds, that give rise to carbonyl group vibrations very close to those observed in pure forms of the β secondary structures.34 As can be seen from the comparison of the spectra (Figure 2B), formation of the LHCII monolayer is associated with pronounced modification of the aggregation status. The enhanced spectral components visible in the low;as well as in the high; wavenumber regions of the amide I band, of the monolayer deposited at the surface pressure that assures trimeric organization of LHCII (15 mN/m), are indicative of efficient formation of intermolecular links based on hydrogen bonds. The fact that those bands are not observed in the spectrum of the monolayer composed of monomeric LHCII suggests that the hydrogen bonds considered can also be involved in stabilization of the trimeric organization of the protein. Formation of such bonds has been implied on the basis of the X-ray crystallography of the complex.11 The difference spectrum (Figure 2B) also shows a relatively intense band, in the low-wavenumber spectral region (centered at 1602 cm-1), which can be attributed to an aggregated structure. Despite the fact that the LHCII preparation examined exclusively contained an aggregated form of the protein (as a precipitate), this band was observed as particularly intense in the spectrum of the monolayer containing trimeric complexes, and it is very likely that it reflects specific molecular organization of LHCII. Assuming that this band represents formally a β-sheet structure in which the amide transition dipole forms an average angle θ = 90 with the main molecular axis,25,34 one can determine the orientation of the peptide fragment which gives rise to the aggregation band. Figure 3 presents the IR absorption spectra in the amide I band, recorded with polarized light. The dichroic ratio corresponding to the aggregation band and calculated on the basis of integration of the Gaussian components centered at 1609 cm-1 (R = A /A^ = 1.960) allowed determination of the angle γ = 67 with respect to the normal axis to the monolayer, as the average orientation of the polypeptide fragment involved in intermolecular interaction between the LHCII trimers. This orientation is clearly larger than the magic angle and implies involvement of the minor protein fragments (e.g., the R-helix D, N-terminal peptide or the interhelical loops) tilted more than the principal helical fragments (R-helix C, ca. 0, R-helices A and B, ca. 45). Linear dichroism-based analysis of another aggregation-related band, centered at 1624 cm-1, leads to the dichroic ratio R = 1.947, which corresponds to the orientation angle γ = 66. As discussed above, it is very likely that this band represents intermolecular interactions between the trimerforming monomers. The linear dichroism analysis of the principal spectral component at 1646 cm-1, attributed to R-helices, gives a mean orientation γ = 55 (R = A /A^ = 1.60, θ = 30 25). (33) Liu, C.; Zhang, Y.; Cao, D.; He, Y.; Kuang, T.; Yang, C. J. Biol. Chem. 2008, 283, 487. (34) Barth, A.; Zscherp, C. Q. Rev. Biophys. 2002, 35, 369.

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Figure 3. Infrared absorption spectra in the amide I region of LHCII in the monomolecular layer deposited by means of the Langmuir-Blodgett technique to ZnSe monocrystal, at the surface pressure 15 mN/m, recorded with the electric vector of incident beam polarized perpendicular (A) and parallel (B) with respect to the plane of incidence. The spectra are presented along with components of Gaussian deconvolution.

The specific LHCII trimer organization in the monolayers gives rise to the excitonic interactions between the protein-embedded pigments, demonstrated at 77 K by the low-energy chlorophyll a fluorescence emission band (the maximum at 700 nm) in addition to the band centered at 680 nm (see Figure 4), recorded also in the case of the trimeric complexes.9,11,35 This particular low-energy excitonic band has been interpreted to have the same spectral origin as the band recorded in this spectral region (at 691 nm), in the oligomerized LHCII, by means of the RLS (resonance light scattering) spectroscopy.9 Interestingly, this long-wavelength emission band is exclusively observed at low temperature. This suggests that either a cooling-induced transition in the LHCII organization in the monolayer takes place, which results in the formation of the low-energy excitonic energy level(s) responsible for light emission, or this energy level is already present at higher temperatures but the yield of fluorescence emission is very low under such conditions. The same phenomenon is observed in the case of bulk oligomeric forms of LHCII in which the corresponding RLS band is also observed at room temperature.9 This suggests that under room temperature conditions thermal energy dissipation competes successfully with fluorescence for de-excitation via this particular energy state. On the other hand, possible low temperature induced structural transformations of the system, which can possibly change the proportion between the (35) Horton, P.; Ruban, A. V.; Rees, D.; Pascal, A. A.; Noctor, G.; Young, A. J. FEBS Lett. 1991, 292, 1.

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Figure 4. Fluorescence emission spectra of LHCII monolayer deposited to nonfluorescent glass slide by means of the Langmuir-Blodgett technique, at a surface pressure of 15 mN/m. The spectra were corrected by subtraction of the background and normalized at the maximum of the 680 nm band. Excitation at 440 nm, excitation slit 10 nm, emission slit 5 nm. Spectra recorded at 293 K (solid line) and at 77 K (dashed line). The fluorescence emission spectra of the LHCII LB film deposited at 35 mN/m are presented as Supporting Information (Figure S1).

Figure 6. Fluorescence excitation spectra of the LHCII monolayer deposited to a glass slide by means of the Langmuir-Blodgett technique, at a surface pressure of 15 mN/m. Spectra were recorded at 293 K ( panel A) and at 77 K ( panel B). Excitation slit 5 nm, emission slit 10 nm; emission was detected at 680 nm (solid line) and at 700 nm (dashed line). Inset shows the spectra with intensity magnified 10-fold.

Figure 5. Infrared absorption spectra in the amide I and II region of LHCII in the monomolecular layer deposited by means of the Langmuir-Blodgett technique to ZnSe monocrystal, at the surface pressure 15 mN/m, recorded at 293 K (solid line) and at 77 K (dashed line). Panel B shows difference spectra: recorded at 77 K minus recorded at 293 K.

de-excitation channels, cannot be excluded. Figure 5 presents the IR absorption spectra, recorded in the amide I and II regions, at room temperature and at 77 K. The difference spectrum 9388 DOI: 10.1021/la900630a

(Figure 5B) shows pronounced temperature-related changes in the molecular organization of LHCII in the monomolecular film. The absorption bands, corresponding to the tightly bound and coisolated with LHCII lipid molecules are practically not altered. This is in regard to the band assigned to the ester carbonyl stretching vibrations at 1735 cm-1 and the scissoring deformation vibrations of CH2 groups of alkyl chains, between 1450 and 1480 cm-1. In the amide I spectral region, the observed, low temperature induced effects are the decreased intensity of the relatively broad band at 1602 cm-1, characteristic of the formation of the specific LHCII network stabilized by the intertrimer hydrogen bonds (see Figure 2B), and concomitant increase in the intensity of the bands attributed to R-helix (1652 cm-1) and to aggregated structures: bands that have to be assigned formally to parallel (1630 cm-1) antiparallel β-sheet (1698 cm-1). The most striking temperature-induced spectral effect is the appearance of the series of bands in the amide II region, at 1576 cm-1, 1558 cm-1, 1540 cm-1, 1522 cm-1, 1506 cm-1, and 1488 cm-1. Interestingly, all those bands are almost perfectly separated by 18 cm-1, which means that we are dealing with the band progression. Owing to the fact that the progression extends the typical amide II spectral region24,25 and that such pronounced effects are not observed in the amide I region, it seems very unlikely that the spectral effects observed in the amide II region are entirely related to the protein component of LHCII. Actually, the band progression observed in the difference spectrum corresponds very well to the band progression typical of the CdC stretching vibrations in the conjugated double bond system of polyenes.36,37 LHCII is (36) Del Zoppo, M.; Bianco, A.; Zerbi, G. Synth. Met. 2003, 139, 881. (37) Lee, J. Y.; Lee, S. J.; Kim, K. S. J. Chem. Phys. 1997, 107, 4112.

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Figure 7. Original fluorescence decay curves (blue trace) of the monolayer deposited to nonfluorescent glass slide by means of the Langmuir-Blodgett technique, at a surface pressure of 15 mN/m (A), 30 mN/m (B), and of LHCII preparation used to prepare monolayers, suspended in the buffer (C). Excitation at 470 nm, emission at 690 nm. The red trace shows the decay curve of the lamp. Multiexponential fit is shown with black line. Lifetime components fitted are presented in the insets to each panel.

a protein that binds a high number of photosynthetic pigments, both chlorophylls and xanthophylls possessing a large number of conjugated CdC bonds. The fact that the IR absorption spectra in the amide II region, of photosynthetic antenna proteins, are not dominated by the bands associated with the CdC stretching vibrations, is primarily due to the fact that these modes are not associated with pronounced dipole transition changes and they are usually silent in the absorption spectroscopy.36,37 Moreover, binding of the pigment molecules to the protein residues results in coupling of the chromophore vibration modes with the vibrations of the protein. In the case of the polyene substitution with polar end groups, the CdC skeletal modes, usually IR-silent, became very active.36 This effect is particularly pronounced upon unsymmetrical substitution of the polyene chain38 (e.g., observed in the case of neoxanthin). Linear dichroism analysis of the band centered at 1558 cm-1 leads to the dichroic ratio R = 2.696 and reveals the average orientation of the polyene molecular director γ = 38 (at 77 K, R = 3.747 and γ = 30) with respect to the normal axis to the plane of the layer, under the assumption that the transition dipole related to the CdC stretching is tilted by the angle θ = 24 with respect to the axis along the polyene direction (such an angle has been determined in the FTIR linear dichroism study of the heptaene chain in the amphotericin B molecule).26 The mean orientation angle found can represent the LHCIIbound xanthophylls, but involvement of the CdC vibrational modes of chlorophylls is also possible and may not be excluded. The fact that upon transition to 77 K this band gains intensity can be interpreted in terms of at least partial uncoupling of the skeletal vibrations of the pigments and the protein. Protein vibrations are able to absorb thermal dissipation energy from the pigments, and therefore, it is possible that the uncoupling postulated to operate at 77 K is responsible for increased fluorescence yield from the low-energy band (Figure 4). Figure 6 presents fluorescence excitation spectra recorded with emission set at 680 nm and at 700 nm. Fluorescence recorded originates from chlorophyll a, and the main component in the excitation spectra is directly related to the Bx band in the Soret region (at 436 nm). The other bands observed in the spectrum indicate efficient functional electronic energy transfer from chlorophyll b (Bx band at 474 nm) and xanthophylls (the superimposed 0-0 vibronic transition of lutein, neoxanthin, and violaxanthin, maximum at 497 nm). The overall shape of the band is very similar to the chlorophyll a excitation band of the suspension of bulk LHCII, but very clearly, the level of the entire band is very low with respect to the portion of the spectrum corresponding to (38) Del Zoppo, M.; Castiglioni, C.; Zuliani, P.; Razelli, A.; Tommasini, M.; Zerbi, G.; Blanchard-Desce, M. J. Appl. Polym. Sci. 1998, 70.

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Figure 8. Spectrum of the average lifetime of LHCII preparation used to prepare monolayers (marked LHCII) and of LHCII in monomolecular layers deposited by means of the LangmuirBlodgett technique to nonfluorescent glass, at the surface pressures 15 and 35 mN/m, indicated. The intensity average lifetimes were calculated on the basis of decay traces and analyses as presented in Figure 7 according to the formula hτ = Σi fiτi; fi = (Riτi)/(ΣiRiτi). In the figure, the results of one set of the experiments are presented, but very similar results have been obtained for another set of experiments.

direct excitation of the Q region of chlorophylls (above 550 nm). This indicates that, in the LHCII trimers organized in the monolayer, the electronic excitations related to the light absorption in the Soret region are partially quenched. The lower intensity of the fluorescence excitation band in the Soret region, relative to the excitation intensity at 600 nm, indicates that the excitation quenching is more efficient at room temperature than at 77 K. This observation corresponds to the concept presented above that transfer of LHCII to low temperature can limit the mechanism of thermal energy dissipation. Interestingly, the effect of selective excitation quenching in the Soret band has not been observed in the LHCII monolayers deposited at 35 mN/m (see Supporting Information Figure S2). The exact mechanism of quenching remains to be solved. Efficient excitation quenching in the Soret region at room temperatures coincides with fluorescence quenching from the low-energy excitonic band (691 nm), and therefore, it is possible that both effects are directly related and that both quenching mechanisms are based on the same specific organization patterns. It is worth mentioning that, in the case of DOI: 10.1021/la900630a

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Figure 9. (A) FLIM image of the 1  1 μm2 area of the LHCII monolayer deposited to the mica slide by means of the Langmuir-Blodgett technique, at a surface pressure of 15 mN/m and (B) average lifetime distribution. The 5.1  5.1 μm2 area of the sample was scanned with a 512  512 pixel resolution. The FLIM image of the LHCII LB film deposited at 35 mN/m is presented as Supporting Information (Figure S3).

the detergent-soluble LHCII preparation, the low-energy excitonic band is neither visible in the RLS nor in the 77 K fluorescence spectra. Contrarily, in the case of the samples composed of the bulk aggregated structures of LHCII such a band can be detected at 691 nm by means of the RLS technique and at 700 nm by means of 77 K fluorescence emission spectroscopy.9 Possible creation of the singlet excitation quenching centers in LHCII organized in monomolecular layers have been analyzed with application of the time-resolved fluorescence technique. Figure 7 presents exemplary fluorescence decay analysis, corresponding to fluorescence emission at 690 nm, and Figure 8 presents the average fluorescence lifetime spectra of the LHCII preparation used to deposit monolayers and of the monomolecular films of LHCII deposited at 15 mN/m (organized trimers) and at 30 mN/m (organized monomers). The lifetime components found (see Figure 7) are very close to those reported for pure LHCII in different organization forms.39 The LHCII preparation shows a certain decrease in average lifetime at wavelengths below 670 nm, but this effect is particularly pronounced in the monomolecular films. In the case of the specific organization of LHCII trimers (monolayer deposited at 15 mN/m), the minimum of the band appears at 690 nm, which corresponds to the excitonic band observed in bulk oligomeric structures formed with the LHCII trimers.9 The local minimum is also observed at 680 nm, in both the monolayers containing structures formed with monomeric and trimeric antenna complexes (Figure 8). This means that in both types of samples formation of spectral forms which give rise to such fluorescence decay components is conserved. A longwavelength tail appears in the average lifetime spectrum representing the structures formed in the monomolecular film at 15 mN/m. Such a tail corresponds to a very clear fluorescence emission shoulder in the region of 720 nm, in the 77 K fluorescence emission spectrum (Figure 4). Such long-wavelength emission spectral forms in LHCII have been attributed to the charge transfer state of two excitonically coupled chlorophyll molecules, each one embedded in neighboring LHCII trimer within the oligomeric structure.40

Figure 9 presents fluorescence lifetime imaging microscopy (FLIM) analysis of the 1  1 μm2 area of the LHCII monolayer composed of organized trimers (deposited at 15 mN/m). As can be seen, the lifetime distribution is not homogeneous over the presented monolayer fragment: mostly, one can detect the relatively short lifetime components, between 0.2 and 0.4 ns, which correspond to the time-resolved spectrum presented in Figure 8. Interestingly, the lifetime component of 0.4 ns has been reported and discussed as typical of the non-photochemical fluorescence quenching in thylakoid membranes41 and in isolated LHCII.40 As could be expected, the long-lifetime components (>0.4 ns) are representative of the molecular structures that are, in most cases, separated one from another (red dots). In contrast, the molecular structures characterized by the short-lifetime components (blue dots) are typically composed of associated substructures and chains. Interestingly, numerous structures characterized by the lifetime between 0.25 and 0.35 ns (green dots) are organized within the closed suprastructures of different shapes and sizes. A spatial arrangement would make such structures extremely attractive in terms of the excitation energy transfer over long molecular distances in the chloroplast membranes. A relative contrast between the neighboring LHCII structures characterized by very short and relatively long lifetimes makes it possible to distinguish such supra-organization forms, but obvious and general limitations of the optical microscopy make it impossible to analyze them precisely. In order to examine organization of the LHCII monolayers at the molecular level, we have applied the atomic force microscopy (AFM) imaging technique. Figure 10A,B presents an AFM image of the 300  300 nm2 fragment of the LHCII monolayer. The bright dots (∼10 nm in diameter) correspond to LHCII trimers. On the other hand, the areas can be observed in the AFM images, which are more homogeneous, without well-resolved trimeric substructures. Interestingly, some structures observed in the AFM-based topography of the monolayer resemble the hexagonal molecular arrangement in the planar crystals of LHCII, reported recently by Barros et al.42 Such a similarity implies molecular organization of the monolayer, in which certain

(39) van Oort, B.; van Hoek, A.; Ruban, A. V.; van Amerongen, H. FEBS Lett. 2007, 581, 3528. (40) Miloslavina, Y.; Wehner, A.; Lambrev, P. H.; Wientjes, E.; Reus, M.; Garab, G.; Croce, R.; Holzwarth, A. R. FEBS Lett. 2008, 582, 3625.

(41) Gilmore, A. M.; Hazlett, T. L.; Govindjee Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 2273. (42) Barros, T.; Royant, A.; Standfuss, J.; Dreuw, A.; Kuhlbrandt, W. EMBO J. 2009, 28, 298.

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Figure 10. AFM images of Langmuir-Blodgett films of LHCII deposited to mica, 300  300 nm2 area top view, z-range 1.6 nm: deposited at

35 mN/m (A), deposited at 15 mN/m (B), and three-dimensional view of 50  50 nm area of the monolayer deposited at 15 mN/m, division in z axis 0.5 nm (C).

neighboring LHCII trimers are oriented antiparallel one to each other. Thorough analysis of several images, including the one presented, let us conclude that the trimers are able to form in the monolayer closed structures of different sizes. The presence of the smallest, ring-like, structures can be traced in the image by appearance of the dark dots in the center. Appearance of such ring-like structures in the LHCII monolayers has been reported previously by Kernen and co-workers.18 Figure 10C presents such a structure composed of five trimers.

Conclusions The results of the experiments presented in this study show that LHCII in the trimeric form is able to form specific, lateral suprastructures stabilized by intertrimer hydrogen bonds. Most probably, the minor helix D or interhelix loops are involved in creation of such links. Specific organization of LHCII trimers is associated with creation of the low-energy singlet excitation quenching centers. The structural organization forms observed

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can be potentially relevant for understanding molecular mechanisms operating in the photosynthetic apparatus to ensure efficient, long-distance excitation transfer and quenching of excess energy under light stress conditions. Acknowledgment. This research was financed by the Ministry of Science and Higher Education of Poland from the funds for science in the years 2008-2011 within the research project N N303 285034. R.L. acknowledges the postdoctoral fellowship from the Ministry of Science and Higher Education of Poland (grant no. 17/MOB/2007/0). The BIONAN network is also acknowledged for financial support. Supporting Information Available: Fluorescence excitation and emission spectra and the FLIM image recorded from the LHCII monolayers deposited at 35 mN/m. This material is available free of charge via the Internet at http://pubs.acs.org.

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