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A Key Role of Xanthophylls that are not Embedded in Proteins in Regulation of the Photosynthetic Antenna Function in Plants, Revealed by Monomolecular Layer Studies Renata Welc, Rafal Luchowski, Wojciech Grudzinski, Micha# Puzio, Karol Sowinski, and Wieslaw I. Gruszecki J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b10393 • Publication Date (Web): 02 Dec 2016 Downloaded from http://pubs.acs.org on December 5, 2016
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ABSTRACT: The main physiological function of LHCII (Light-harvesting pigment-protein complex of Photosystem II), the largest photosynthetic antenna complex of plants, is absorption of light quanta and transfer of excitation energy toward the reaction centers, to drive photosynthesis. However, under strong illumination, the photosynthetic apparatus faces the danger of photo-degradation and therefore excitations in LHCII have to be down-regulated, e.g. via thermal energy dissipation. One of the elements of the regulatory system, operating in the photosynthetic apparatus under light stress conditions, is a conversion of violaxanthin, the xanthophyll present under low light, to zeaxanthin, accumulated under strong light. In the present study an effect of violaxanthin and zeaxanthin on the molecular organization and the photophysical properties of LHCII was studied in a monomolecular layer system with application of molecular imaging (Atomic Force Microscopy, Fluorescence Lifetime Imaging Microscopy) and spectroscopy (UV-Vis absorption, FTIR, fluorescence spectroscopy) techniques. The results of the experiments show that violaxanthin promotes formation of supramolecular LHCII structures preventing dissipative excitation quenching while zeaxanthin is involved in formation of excitonic energy states able to quench chlorophyll excitations both in the higher (B states) and lower (Q states) energy levels. The results point to a strategic role of xanthophylls that are not embedded in a protein environment, in regulation of the photosynthetic light harvesting activity in plants.
2
INTRODUCTION LHCII (Light-harvesting pigment-protein complex of Photosystem II) is a major component of the photosynthetic antenna system in plants.1,
2, 3, 4, 5
The main
physiological role of this pigment-protein complex is absorption of light quanta and transfer of electronic excitations towards the reaction centers. On the other hand, the exceptionally high effectiveness of the complex in light harvesting may be considered as acting to the detriment of the photosynthetic apparatus, under strong light conditions, increasing to the risk of photo-oxidation.6 Therefore, a protection against photodegradation may be considered vital to photosynthesizing organisms. There are several mechanisms reported, acting as a self-defense against overexcitation-induced damage, operating at the level of antenna complexes.6,
7, 8, 9, 10
Interestingly, the one of the
mechanism involved in photoprotection is based upon the structural reorganization of the photosynthetic antenna protein system, which is tightly related to presence of the xanthophyll cycle pigments violaxanthin (Vio) and zeaxanthin (Zea).11,
12
Vio is a
carotenoid present in the photosynthetic apparatus of plants under low light conditions and is enzymatically converted to Zea, in the xanthophyll cycle.13,
14, 15, 16, 17
In the
present study we address the problems of regulatory mechanisms underlying photoprotective energy dissipation in the supramolecular structures of the LHCII, dependent and modulated by presence of the xanthophyll cycle pigments Vio and Zea. A monomolecular layer approach, combined with several molecular spectroscopy and imaging techniques, was applied to get insight into the molecular and photo-physical mechanisms underlying such a regulatory activity. The monomolecular layer system has been shown to provide a suitable model for study of the molecular organization and
3
structural features of LHCII, in which the complex retains its native structure and functional properties, manifested by efficient excitation energy transfer.18, 19, 20, 21
METHODS LHCII and Xanthophyll Isolation and Purification. Light-harvesting pigmentprotein complex LHCII was isolated from fresh spinach (Spinacia oleracea L.) leaves according to the method developed by Krupa et al.
22
. Purity of the preparations has
been controlled with application of spectroscopic and electrophoretic techniques.23,
24
All-trans violaxanthin and all-trans zeaxanthin were isolated from Narcissus jonquilla L. flowers and Lycium barbarum fruits, respectively, according to the procedures described previously.25,
26
Directly before use, xanthophylls were purified by means of
the HPLC technique, with application of a C-18 coated, phase-reversed column (YMC GmbH, Germany, length 250 mm, internal diameter 4.6 mm). A mixture of acetonitrile:methanol:water (72:8:3, by volume) was used as mobile phase and elution rate was 0.8 ml/min. Spectral characteristics of Zea and Vio purified for the purpose of the present study are in perfect agreement with the characteristics reported in the literature.27 Monomolecular
Layers.
Different
types
of
monolayers
were
formed,
compressed and analyzed with application of a commercial Minitrough system from KSV Instruments (Finland). Monolayers were formed at the air-water interface. Ultrapure water, used as subphase, was prepared in a Milli-Q system from MerckMillipore. Water specific resistivity was 18 M cm. Monolayers of pure LHCII were deposited from a 25% (by volume) isopropanol solution in tricine buffer.19,
4
20
Such a
method yields the formation of a monolayer composed of intact and functional LHCII complexes.28 Monolayers of pure xanthophylls were deposited from pigment solutions in ethanol:hexane 1:6 (v:v) solvent mixture. Specific molecular areas of pure Vio and Zea in monolayers (41 ± 8 Å2 and 40 ± 7 Å2 respectively, see Supporting Information Figure S1) correspond very well to the values reported previously.29,
30, 31
In order to form
mixed, two-component systems of LHCII and xanthophylls, Vio or Zea solution was deposited first. After time needed for evaporation of solvents (15 min), LHCII was deposited at the interface. The molar ratio of an exogenous xanthophyll to LHCII was 1:1. Monolayers were transferred to a solid support by means of the Langmuir-Blodgett (L-B) technique, at a constant, computer-stabilized surface pressure of 20 mN/m and a rate of compression set to 750 mm2/min. Spectroscopic measurements of monolayers were carried out directly after deposition. All experiments were performed at 23 ± 1 oC temperature. Spectroscopic Analyses. Infrared absorption spectra were recorded with a FTIR Nicolet iS50 spectrometer (Thermo Scientific, USA), equipped with a horizontal attenuated total reflection cell (ATR). A high sensitivity MCT detector cooled by liquid nitrogen was used. Monolayers to be measured were deposited to a ZnSe crystal by means of the Langmuir-Blodgett technique. Absorption spectra were collected in the region between 4000 and 600 cm-1 with a 4 cm-1 resolution. One hour before and during measurements the instrument was purged with argon. UV-Vis absorption spectra were recorded with application of a Cary50 spectrophotometer (Varian, Australia). Steady-state fluorescence emission spectra were recorded with a Cary Eclipse spectrofluorometer (Varian, Australia). The excitation wavelength was set at 470 nm and the emission spectra were recorded in the range of 600–850 nm. The 5
measurements were carried out at liquid nitrogen temperature (77 K). Fluorescence excitation spectra were recorded with application of a FS5 spectrofluorometer (Edinburgh Instruments, UK). Emission was set at 695 nm and spectra were recorded in the range of 450-690 nm. Fluorescence spectra were recorded with bandpass corresponding to the widths of 5 nm, both in the excitation and emission beams. Fluorescence spectra were corrected for lamp intensity and detector sensitivity. AFM imaging. AFM (Atomic Force Microscopy) imaging of monolayers was performed with application of a NanoWizard 3 microscopic system (JPK, Germany) in the noncontact mode. Monolayers were deposited to freshly cleaved mica sheets by means of the Langmuir-Blodgett technique. Super Sharp Silicon cantilevers (force constant: 21-98 N/m, resonance frequency: 146-236 kHz) were used. FLIM Imaging. FLIM (Fluorescence Lifetime Imaging Microscopy) imaging was performed with application of a confocal MicroTime 200 (PicoQuant, Germany) system coupled to an inverted microscope OLYMPUS IX71. Monolayers were deposited to nonfluorescent glass slides (24x50 mm, #1, Menzel-Glaser). The laser power at a sample was 0.7 μW. The laser excitation wavelength was 470 nm and repetition frequency was 20 MHz. The lifetime resolution was set to 16 ps. Photons were collected with a 60x water immersed objective (NA 1.2, OLYMPUS UPlanAPO). A single focal plane was selected with a pinhole diameter of 75 μm. Scattered light was removed using a dichroic ZT473RDC XT and 690/70 band pass filters, both from ANALYSENTECHNIK (Germany). Measurement results were analyzed using a SymPhoTime 64 software from PicoQuant (Germany).
6
Spectroscopic measurements and imaging were recorded for several times, from the different samples representing all kind of systems (LHCII, LHCII+Vio, LHCII+Zea), and the results were highly reproducible.
RESULTS Monomolecular Layers at the Air-Water Interface. Figure 1 presents isotherms of compression of monomolecular layers formed at the air-water interface with LHCII and the two component systems comprising LHCII and exogenous Vio or Zea (in a molar fraction 1 exogenous xanthophyll per 1 monomer of LHCII). As can be seen, the limiting molecular area of LHCII, determined in a single-component system, Ao=1530 ± 164 Å2 corresponds very well to the values determined in the previous studies, 1433 Å2
20
and 1395 Å2
19
. A monomer of LHCII has roughly an elliptical cross-
section, defined by the 30 Å x 60 Å axes,1,
2
which can give rise to limiting areas
approaching 1414 Å2 in a most packed system. The presence of exogenous xanthophylls in monomolecular layers formed with LHCII shifts the limiting area parameter towards higher values by ca. 1000 Å2 (Figure 1). Such a shift is much higher than can be expected taking into consideration the fact that one exogenous xanthophyll was added per one LHCII monomer and that the limiting area of molecules of Vio and Zea, in the single component monolayers, were close to 40 Å2 (see the Supplementary Information Figure S1). Such a pronounced over-additivity can be interpreted in terms of supramolecular organization of LHCII, in the presence of Vio and Zea, which generates substantial fraction of unoccupied surface area in a monomolecular layer. Motifs of the possible molecular arrangements in such systems, giving rise to the surface area over-
7
additivity observed, are proposed in Figure 2. In the model presented, the molecules of exogenous Vio and Zea interact with LHCII via hydrogen bonding between the hydroxyl groups located at the 3 and 3’ positions in Vio, Zea and the hydroxyl groups of the protein-bound neoxanthin (Neo). The model corresponds well to the topographies of monomolecular layers deposited to the mica surface, by means of the LangmuirBlodgett technique and analyzed with application of AFM (see Figure 3 and the Supporting Information Figure S2). As can be seen, the films formed with LHCII and exogenous Vio and Zea contain several pore-like structures. Interestingly, the diameters of the pores observed in the LHCII+Zea system are clearly higher as compared to the pore diameters observed in the LHCII+Vio system. Certain defects in the monolayer structure, observed also in the case of mono-component LHCII monolayers as irregular black holes, are created, most probably, during film deposition. Otherwise, the LHCII monolayer is relatively smooth and regular. It is possible that molecules of LHCII adopt two different orientations with respect to the interface (with C terminus or N terminus contacting the water subphase) in a mono-component system. On the other hand, it is very likely that interaction of the protein to exogenous xanthophylls, shown to affect considerably a monolayer organization, favors the orientation in which the xanthophylls linking neighboring LHCII trimers are located at the air-water interface. Interestingly, the presence of exogenous xanthophylls, and in particular Zea, substantially increases values of the compression modulus (see Figure 4). A pronounced increase in rigidity of the system containing exogenous Zea may be a direct consequence of molecular organization of the system, stabilized by directional hydrogen bonds.
8
Spectroscopic Analysis of Langmuir-Blodgett Films. In order to get insight into consequences of supramolecular organization of LHCII in the presence of exogenous xanthophylls, monolayers formed at the air-water interface were deposited to a solid support and examined with application of molecular spectroscopy techniques. Figure 5 presents comparison of IR absorption spectra recorded from the films formed with pure LHCII and LHCII containing exogenous Vio and Zea. As can be seen, the relatively intensive band in the spectral region between 1620-1640 cm-1, representing formation of lateral aggregated structures by LHCII,19, 32 is not present in the case of the xanthophyll-modified systems. This is a clear demonstration that the presence of exogenous xanthophylls in the system hinders protein-protein interactions leading to LHCII aggregation. The same conclusion can be drown based on the comparative analysis of the low-temperature chlorophyll a (Chl a) fluorescence emission spectra in LHCII and LHCII+Vio or LHCII+Zea systems (see Figure 6). The relatively intense band at 700 nm, representing aggregated forms of the protein
12, 33, 34
is particularly
pronounced in the system composed of pure LHCII. On the other hand, the band with the maximum in the region of 680 nm, representing a non-aggregated protein fraction, is relatively intensive in the systems containing exogenous Vio and Zea. Figure 7 presents absorption spectra of L-B films, recorded in the UV-Vis spectral region. The main observation from the comparison is a broadening of the absorption band in the Soret region in the spectrum recorded from the LHCII+Zea system. The difference spectrum shows appearance of a relatively strong band in the region between 500 and 600 nm. Such a broad band does not match the absorption spectrum of isolated Zea (between 400 and 500 nm
35
) and rather represents additionally excitonic interactions of
molecules of an exogenous xanthophyll with pigments embedded in LHCII.35, 36, 37 The 9
fact that the excitonic band of Zea is shifted towards greater wavelengths implies formation of J-type structures.36,
37
The difference spectrum of the LHCII+Vio and
LHCII+Zea systems (Figure 7, lower panel) was additionally analyzed in the “green” spectral region, based on Gaussian deconvolution. The two spectral components resolved can be interpreted as representing the Zea minus Vio difference spectrum (maximum at 507 nm
38
) and the excitonic band (539 nm). Very close, two-component
spectrum also was recorded in intact leaves subjected to illumination, and correlated to zeaxanthin accumulation.39, 40, 41 Interestingly, only the first component, centered at 507 nm and assigned to the Zea minus Vio difference spectrum, was observed as accompanying Vio deepoxidation in the plants lacking LHCII: in etiolated leaves42 and in leaves of intermittent-light-grown plants.43 Such a result corroborates with the interpretation according to which the excitonic band at ca. 530 nm is directly related to the interaction of Zea with LHCII. The presence of two components in the "green" spectral region of the difference spectrum is indicative of at least two fractions of Zea in the samples, of which only one involved in excitonic interactions giving rise to pronounced bathochromic shift. A similar, heterogeneous distribution of Zea can be expected in intact photosynthetic apparatus, as can be concluded based on complexity of the absorption spectra recorded from intact leaves
39, 40, 41
. Interestingly, the
differences can also be observed in the red spectral region (Figure 7, lower panel), despite normalization at the Qy maximum. The pronounced spectral effects in the red spectral region, associated with Zea presence in the system, comprise slight bathochromic shift of the Qy band of Chl a, strong decrease in intensity of the Qy band of Chl b and substantial increase in intensity between 700 nm and 750 nm. It is possible that the new, most red-shifted band represents excitonic interactions of the Neo-Zea 10
interacting pair of xanthophylls with a near-by Chl b molecule(s) (e.g. Chl b 609). This particular band could be responsible for the long-wavelength fluorescence emission band, present in the LHCII+Zea system, with the maximum at 730 nm (see Figure 6). Importantly, very strong excitonic coupling between Chl b and Neo has been deduced based on the results obtained with application of Stark effect spectroscopy. 44 The assignment of the new, low-energy band at 730 nm, to the excitonic interaction of Chl b has also a support from the analysis of fluorescence anisotropy distribution (see the Supporting Information Figure S3). The fact that excitation at 470 nm results in population of the Bx energy level of Chl b while fluorescence emission in LHCII originates from the Qy energy level of Chl a is associated with fluorescence anisotropy distribution near 0. This is particularly pronounced in the case of LHCII+Vio system, owing to formation of supramolecular structures promoting efficient, long range excitation energy transfer pathways (see Figure S2). Importantly, the fluorescence anisotropy distribution recorded for the LHCII+Zea systems contains a component shifted distinctly towards positive values. Such a component can be expected in the case of a direct radiative deexcitation from the low excitonic energy level of Chl b molecule involved in excitonic interactions with xanthophylls (without energy transfer to Chl a and internal conversion between the states with orthogonal transition dipoles). Figure 8 presents comparison of Chl a fluorescence excitation spectra in L-B films deposited from monolayers composed of LHCII, pure and in the presence of exogenous Vio and Zea. The spectra were normalized in the Qy maximum of Chl a. As can be seen, all the spectra recorded from different systems match each other very well, in the long-wavelength spectral region. This is in sharp contrast to the short-wavelength spectral region (see Figure 8). The relative intensity of the Soret band, in the films 11
containing additional xanthophylls, is strongly decreased (by almost a factor of 2 in the case of LHCII+Zea as compared to pure LHCII). Such a decrease cannot be explained in terms of a light filtering effect by additional xanthophylls owing to the fact that molecules of exogenous Vio and Zea are present in the same plane as LHCII in the monomolecular layers (as can be concluded based on analysis of the isotherms of compression, Figure 1). Furthermore, the magnitude of the effect observed cannot be justified by the presence of one additional carotenoid pigment per several endogenous chlorophylls and xanthophylls. The most plausible explanation of the decrease in intensity of the Soret band, relative to the Qy band, associated with the presence of additional xanthophylls, could be selective quenching of excitations of higher electronic levels via energy transfer to excitonic energy levels overlapping the Soret band, in the case of the LHCII+Vio system, or located lower on energy scale, in the case of LHCII+Zea (see Figure 7). In the same samples, the fluorescence lifetime analysis shows that the lowest energy levels of Chl a in LHCII, responsible for fluorescence emission, are not quenched by additional xanthophylls (Figure 9). Moreover, the average fluorescence lifetimes are greater in the LHCII+Vio and LHCII+Zea systems as compared to the L-B film formed with pure LHCII, confirming the aggregation-preventing effect of exogenous xanthophylls. Importantly, the level of the xanthophyll cycle pigments has been correlated with the Chl a fluorescence excitation quenching, selectively in the Soret spectral region, in intact leaves of Arabidopsis thaliana.45
DISCUSSION
12
The results of the analyses presented in this report clearly demonstrate that both the xanthophyll cycle pigments, Vio and Zea, modulate molecular organization of the pigment-protein complex LHCII (see Figure 1 and Figure 4). An important consequence of such an effect is a hindrance of the protein self-association that results in excitation quenching (Figure 9). The xanthophyll-related prevention against LHCII aggregation also can be directly concluded from the results of the FTIR analyses (Figure 5) and lowtemperature fluorescence emission spectroscopy (Figure 6). This mechanism seems to be particularly important from the standpoint of an excitation energy transfer optimization and supply to the reaction centers in the photosynthetic apparatus of plants under low light conditions. According to several reports, Vio, which is available for deepoxidation, is present not only bound to the pigment-protein complexes in the thylakoid membranes but also in a relatively mobile fraction.12,
14, 46, 47
Such a Vio
fraction can be involved in stabilization of supramolecular structures of LHCII.12 The concept that Vio can be involved in stabilization of LHCII clusters also has a support from the results of the freeze-fracture electron microscopic analysis of the thylakoid membranes from spinach chloroplasts.10 The analyses have shown that in the darkadapted chloroplasts containing Vio, the LHCII particles were evenly distributed across the fracture face with clear gaps among them.10 In opposite, formation of highly compact LHCII clusters has been found in the thylakoid membranes of the chloroplasts subjected to illumination and containing Zea.10 The results of the experiments carried out in monomolecular layer systems, reported in the present work, demonstrate that in the case of the presence of Zea, a separation between the neighboring LHCII complexes is even higher, in contrast to the natural biomembrane study. Such a discrepancy implies that Zea in the thylakoid membranes is not involved in stabilization 13
of long-range planar structures observed in monomolecular layers. It seems very likely that the presence of the PsbS protein in the thylakoid membranes, which is known to bind selectively Zea molecules and interact with LHCII,48 plays a key role in molecular organization of LHCII in the presence of Zea in vivo. Also, it was shown that Zea induces destabilization of the trimeric organization of LHCII,12 which is a basic structural requirement for the formation of the planar structures imaged in Figure 3 and modeled in Figure 2. On the other hand, also it is very probable that certain elements of the molecular architecture observed in the two-component LHCII+Zea monolayers will appear in the natural systems. This is simply owed to the fact that the same molecular forces are active both in the natural and model systems. One consequence of such a molecular organization, which seems very important from the physiological standpoint, is the formation of Zea complexes with LHCII, which directly result in creation of excitonic bands in the “green” spectral region (Figure 7). A broad band in the absorption spectra recorded from intact leaves, closely resembled the excitonic band presented in Figure 7, has been reported in several studies of intact photosynthetic systems.39, 40, 41 Energy levels responsible for this excitonic band (between 500 and 600 nm) have a potential to quench electronic excitations in the Soret band, as manifested in the Chl a fluorescence excitation spectra (Figure 8). A schematic energy level diagram of the photosynthetic pigments, which comprises such an excitonic energy level, is presented in Figure 10. A direct cause of a pronounced bathochromic shift of the absorption band associated with this excitonic interaction (Figure 7), is most probably a collinearity of the LHCII-bound Neo and exogenous Zea molecules (a structural prerequisite for formation of J-type structures). Literature provides numerous contradictory results concerning a causative relationship between the presence of Zea and excitation quenching in the 14
photosynthetic apparatus of plants. On the one hand, a role of Zea accumulation for photoprotection has been proven based on the studies carried out with the npq1 mutant of Arabidopsis with impaired Vio deepoxidation.41 On the other hand, a direct role of Zea in photoprotective excitation quenching was questioned by the results of the studies carried out on intact maple leaves.
49
The recent results suggest that Zea does not
exchange for Vio in the pigment binding pockets in antenna proteins but acts rather in modulation of organization antenna complexes, which results in creation of quenching sites.11 In light of the findings of the present study, interaction of Zea pool, which is not protein-embedded, with the LHCII-bound Neo, can be responsible for creation of excitonic energy levels able to effectively quench chlorophyll excitations in the higher energy levels, even before relaxation to the Qy state (Figure 10). Moreover, analyses of the low-temperature Chl a fluorescence emission spectra from the LHCII+Zea system reveals formation of a long-wavelength energy level, corresponding to the emission maximum at 730 nm (Figure 6), which can also be responsible for excitation quenching at a level of the Qy state of Chl a.
CONCLUSIONS Analysis of two-component monomolecular layers formed with LHCII and the xanthophyll cycle pigments, violaxanthin and zeaxanthin, reveals specific interactions of both xanthophylls with the protein. The interactions determine molecular organization of the complex and critically influence photophysical properties of the protein-embedded pigments. In the case of violaxanthin, the supramolecular structures formed minimize energy loses and assure efficient, long range excitation energy transfer. In the case of 15
zeaxanthin, the specific interactions to LHCII result in creation of excitonic energy levels able quench excessive excitations directly at the energy levels corresponding to the Soret band. Such a mechanism may be considered vital under light stress conditions. The results point to a key role of LHCII interactions with external xanthophylls in regulation of the photosynthetic antenna function at the molecular level.
ASSOCIATED CONTENT Supporting Information: Isotherms of compression of monolayers formed with pure violaxanthin and zeaxanthin, cross-section analysis of AFM images, analysis of distribution of fluorescence anisotropy in L-B films.
Author Information Corresponding Author *E-mail:
[email protected] Notes The authors declare no competing financial interest.
ACKNOWLEDGEMENTS R.W. acknowledges the National Science Center of Poland (NCN) for financial support within the project UMO-2014/13/N/NZ1/00998. The research was carried out with the 16
equipment purchased thanks to the financial support of the European Regional Development Fund in the framework of the Development of Eastern Poland Operational Programme.
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Figure 1. Surface pressure - mean molecular area isotherms of compression of the monolayers formed with LHCII and LHCII mixed with exogenous violaxanthin and zeaxanthin. The isotherms are presented as a function of mean molecular area per molecules of LHCII both in the mono-component and two-component systems. The straight lines fitted to the linear portions of the isotherms, extrapolated to the zero surface pressure, indicate the specific molecular area per LHCII monomer. The legend gives specific molecular area values as an average from 6 experiments ± S.D.
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Figure 2. Models proposing specific motifs of the molecular packing in the monomolecular layers formed with pure LHCII and two-component systems formed with LHCII and violaxanthin or LHCII and zeaxanthin. Created with application of the YASARA software using the PDB model: 2BHW. In the models of LHCII, only the protein component and neoxanthin are displayed. Molecules of neoxanthin can form intermolecular hydrogen bonds with exogenous violaxanthin and zeaxanthin owing to the fact that only one part of the xanthophyll is embedded in the protein environment while the other end protrudes out of the complex.
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Figure 3. AFM images of 500 x 500 nm areas of the Langmuir-Blodgett films deposited from monomolecular layers formed with pure LHCII and two-component films formed with LHCII with violaxanthin or zeaxanthin. Monolayers were deposited to the surface of mica. Scale bar = 100 nm.
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Figure 4. Compressibility modulus of monolayers formed with pure LHCII, pure xanthophylls and two-component monolayers formed with LHCII with violaxanthin and 𝑑𝜋
LHCII with zeaxanthin. Compressibility Modulus was calculated as: 𝐶𝑠−1 = −𝐴 (𝑑𝐴), where 𝐴 denotes area per molecule and 𝜋 denotes surface pressure (based on ref. 50).
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Figure 5. Infrared absorption spectra of LHCII, in the amide I spectral region, recorded from the Langmuir-Blodgett films deposited from the mono-component and twocomponent monolayers formed with LHCII and LHCII modified with exogenous violaxanthin or zeaxanthin. Monolayers were deposited to the surface of ZnSe crystal. The spectra were normalized at 1651 cm-1 (the maximum of the principal spectral component representing an α-helical secondary structure of the protein).
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Figure 6. Low-temperature (77 K) fluorescence emission spectra recorded from Langmuir-Blodgett films deposited from the monolayers formed with LHCII and LHCII with violaxanthin or with zeaxanthin. The excitation wavelength was set at 470 nm. The spectra were normalized to get the same area beneath each spectrum. Positions of selected bands are marked.
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Figure 7. Absorption spectra recorded from Langmuir-Blodgett films deposited from the monolayers formed with LHCII and LHCII with violaxanthin or with zeaxanthin. The spectra were normalized at the Qy maximum of chlorophyll a. Lower panel presents the difference spectrum calculated by subtraction of the spectrum recorded from the system enriched with violaxanthin from the spectrum recorded from the system enriched with zeaxanthin. The positive band recorded in the “green” spectral region has been
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analyzed in terms of the Gaussian deconvolution based on two components (dashed red lines), centered at 507 nm and 539 nm. The reconvoluted band is displayed by a thin red continuous line.
Figure 8. Comparison of the chlorophyll a fluorescence excitation spectra in LangmuirBlodgett monolayers formed of pure LHCII and LHCII with violaxanthin or zeaxanthin. The spectra were normalized at the Qy maximum of chlorophyll a.
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Figure 9. FLIM images of 10 x 10-μm areas of the Langmuir-Blodgett films deposited from the mono-component and two-component monolayers formed with LHCII and LHCII modified with exogenous violaxanthin or zeaxanthin. Fluorescence decay traces, representing all the photons collected in course of the monolayer imaging, are presented below each image along with the intensity-weighted average lifetime of chlorophyll a fluorescence in each scanned sample. Scale bar = 2 µm.
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Figure 10. Simplified energy level diagram of chlorophylls in LHCII with indicated localization of energy levels of zeaxanthin (Zea) and excitonic energy levels related to interaction between the protein-bound neoxanthin (Neo) and zeaxanthin (Zea). Selected transitions between electronic states are represented by arrows. The transitions related to the presence of new excitonic energy levels are marked in red color. The transitions associated with the main pathways of zeaxanthin-related excitation energy dissipation are marked by the wavy arrows.
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