Article pubs.acs.org/Langmuir
Effect of Protonation on the Secondary Structure and Orientation of Plant Light-Harvesting Complex II Studied by PM-IRRAS Tonya D. Andreeva,*,† Sabine Castano,‡ Sashka Krumova,† Sophie Lecomte,‡ and Stefka G. Taneva† †
Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., bl. 21, 1113 Sofia, Bulgaria ‡ CBMN-Univ. Bordeaux, UMR 5248, Allée Geoffroy Saint Hilaire, 33600 Pessac, France ABSTRACT: The major light-harvesting pigment−protein complex of photosystem II, LHCII, has a crucial role in the distribution of the light energy between the two photosystems, the efficient light capturing and protection of the reaction centers and antennae from overexcitation. In this work direct structural information on the effect of LHCII protonation, which mimics the switch from light-harvesting to photoprotective state of the protein, was revealed by polarization-modulated infrared reflection−absorption spectroscopy (PMIRRAS). PM-IRRAS on LHCII monolayers verified that the native helical structure of the protein is preserved in both partly deprotonated (pH 7.8, LHCII) and protonated (pH 5.2, p-LHCII) states. At low surface pressure, 10 mN/m, the orientation of the α-helices in these two LHCII states is different tilted (θ ≈ 40°) in LHCII and nearly vertical (θ ≈ 90°) in p-LHCII monolayers; the partly deprotonated complex is more hydrophilic than the protonated one and exhibits stronger intertrimer interactions. At higher surface pressure, 30 mN/m, which is typical for biological membranes, the protonation affects neither the secondary structure nor the orientation of the transmembrane α-helices (tilted ∼45° relative to the membrane surface in both LHCII states) but weakens the intermolecular interactions within and/or between the trimers.
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INTRODUCTION The major light-harvesting complex of photosystem II (LHCII) is the most abundant protein in the thylakoid membrane of higher plants and serves as an antennae complex of photosystem II (PSII). Under specific light conditions it is able to laterally migrate in the membrane, energetically connect to photosystem I (PSI), and serve as its antenna.1−3 The crystal structure of LHCII was initially solved by Kühlbrandt et al.4,5 and later on improved by Liu et al.6 to 2.72 Å resolution. The three transmembrane α-helices (A, B, and C) and two short amphiphilic helices (D and E) were found to accommodate eight chlorophyll a and six chlorophyll b molecules, that are vertically arranged in either stroma or lumen facing layer, and three to four xanthophylls.6 As shown by Nussberger et al.,7 phosphatidyl glycerol and digalactosyl diacyl glycerol are specifically associated with LHCII, and the complete delipidation of LHCII results in dissociation of the native trimeric form of the complex into a monomeric one. The trimers are also able to form higher-order aggregated structuresshown to be functionally important and involved in the photoprotective mechanisms that spare the thylakoid membranes damage by reactive oxygen species.8 LHCII is mainly distributed in the stacked (granal) regions of the thylakoid membrane either attached to or detached from the periphery of the PSII complexes, providing high or low excitation flux toward the PSII reaction centers, respectively. These two localizations are believed to correspond to two © XXXX American Chemical Society
functional states of the complexlight-harvesting (captures and directs the excitation energy toward the reaction centers) and photoprotective (dissipates the harmful excess light energy);9−12 the latter was shown to be associated with LHCII aggregation that can be triggered chemically by lowering the pH of the medium.13,14 Loosely stacked lamellar aggregates of LHCII, isolated by mild detergent treatment, as well as LHCII microcrystals have been shown to retain close to the native LHCII organization and to undergo reversible light-induced structural changes.15−18 The high structural flexibility of the LHCII macroaggregates was suggested to have physiological significance in the processes of light adaptation and photoprotection.19 LHCII monolayers on liquid and solid supports are extensively investigated as a suitable model system for the study of the supramolecular organization of the complex under different experimental conditions that are not feasible to perform in the highly complex and crowded (80% protein) thylakoid membrane.20−24 We have recently examined the relations between the lateral LHCII rearrangements and the conformational changes during the molecular switch from lightharvesting to photoprotective state of LHCII by comparing the physicochemical properties of partly deprotonated (LHCII, Received: July 19, 2015 Revised: September 30, 2015
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Figure 1. Surface pressure/mean monomer area (π/A) isotherms (a) and PM-IRRAS spectra of partly deprotonated LHCII (blue) and protonated p-LHCII (red) monolayers at π = 10 mN/m (b) and π = 30 mN/m (c) on a buffer subphase.
Figure 2. Decomposition of the PM-IRRAS spectra of LHCII (left) and p-LHCII (right) monolayers at 10 mN/m (a and b) and 30 mN/m (c and d). Decomposition components of the negative band in p-LHCII spectrum (b) are not presented.
protonation exerted strong effect on the proteins supramolecular organization and led to significant remodeling of the monolayer, making it more heterogeneous as compared to the one characteristic for the partly deprotonated state.25 Here we use the same model system to further examine how the established changes in the macroorganization of the complex triggered by its protonation are associated with changes in the LHCII secondary structure. PM-IRRAS technique was applied to characterize the conformation and orientation of LHCII in monolayers at the air/buffer interface at two protonation states. The monomolecular films were transferred to solid supports, and their hydrophobicity was determined by the contact angle method.
light-harvesting state) and protonated (p-LHCII, photoprotective state) LHCII in Langmuir monolayers.25 We have found a different contour of the π/A isotherms and difference in the hysteresis of the LHCII and p-LHCII monolayers. LHCII monolayers suffered irreversible changes in conformation and macroorganization, whereas p-LHCII monolayers were able to reverse to their original state after compression− decompression cycle. The protonation-induced switch in the LHCII configuration yielded more tightly packed, stable but flexible macroorganization (in terms of reversibility of the compression-induced structural rearrangements) of the complexes. Brewster angle microscopy images of LHCII and pLHCII monolayers unambiguously demonstrated that the B
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intense band, centered at 1651 cm−1, originates from α-helices; the less intense one at 1626 cm−1 and the very weak one at 1689 cm−1 are formally assigned to parallel and antiparallel βsheets, respectively, but later on are attributed to supramolecular LHCII structures;27,30 the band at 1673 cm−1 is assigned to β-turns and loops. The band at 1605 cm−1 can be ascribed to the two short antiparallel strands, found in the lumenal loop of the complex,6 or to the minor α-helices D and E and the interhelical loops.30 An analogous particularly intense band (as in our case) was observed in the spectrum of monolayer composed of trimeric LHCII complexes but was missing in the case of monomeric LHCII, hence reflecting the specific molecular organization of the protein.30 The presence of a peak at 1626 cm−1, which also exists in the Fourier transform infrared (FTIR) spectra of aqueous LHCII solutions,9,26,27 is an indication that spreading of LHCII at the air/buffer interface does not cause unfolding of the protein complex, which would be manifested by modification of its native α-helical structure and production of β-sheets. The fact that this peak is stronger in the PM-IRRAS compared with that in the FTIR spectrum may be due to the fixed LHCII orientation at the interface. The spectra in Figure 1 were analyzed based on the PMIRRAS “surface-selection rule” and simulated spectra of proteins in the spectral region of the amide I and amide II bands at different orientations of α-helices and β-sheets.31,32 According to the “surface-selection rule”, intense positive amide I and much weaker positive amide II bands (the ratio of the intensities of amide I and amide II bands, IAI/IAII ∼6.5) originate from helices lying along the water surface (tilt angle θ = 0°). In contrast, helices perpendicular to the water surface (tilt angle θ = 90°) give rise to a strong negative amide I and strong positive amide II bands (IAI/IAII ≈ −1). For tilted helices with a tilt angle 0° < θ < 90°, the amide I band shows derivative-like shape, and the IAI/IAII ratio is the parameter used to estimate the tilt angle. It is already established that changing the α-helical orientation from surface-aligned to upright corresponds to reduction of the IAI/IAII ratio from 6.5 to −1.31 The spectrum of the partly deprotonated LHCII in Figure 1b reveals that the intensity ratio of amide I and amide II bands, IAI/IAII, is 1.3 (1.4 ± 0.2 for nine spectra recorded, three for each of the three independent LHCII isolations). The transition dipole moment of the amide II band (due to C−N stretching and N−H bending) lies in the plane of the helix, whereas the transition dipole moment of the amide I band (arising from CO stretching) lies out of the plane of the helix, since both amide I and amide II transition dipoles are approximately perpendicular to each other. Considering the “surface-selection rule” of PM-IRRAS at the air/water interface, the ratio of 1.3 indicates that the transition dipole moments of amide I and amide II modes are both preferentially oriented in planes that lie in-between the parallel and perpendicular orientation with respect to the interface plane. Compared with the calculated PM-IRRAS spectra of an α-helix, we can estimate that at low surface pressure LHCII is mainly composed of tilted α-helices with an average tilt angle θ ≈ 40 ± 2° with respect to the interface. This angle approximates the one determined by crystallographic studies revealing that the LHCII monomer has three hydrophobic transmembrane helicesA and B (inclined at an angle of ∼30° with respect to the membrane plane) and C (nearly perpendicular to it) that are connected by hydrophilic loopsand two short amphiphilic helices (D and E) exposed
RESULTS AND DISCUSSION The π/A isotherms of both protonated and partly deprotonated LHCII monolayers (Figure 1a) show two (low- and highsurface pressure) regions with different slopes, suggesting that conformational and/or orientational changes take place as a result of the monolayer compression (for detailed discussion, see ref 25). We have previously established the optimal conditions for the formation of stable monolayers and have found that the protonated and partly deprotonated LHCII exhibit distinct supramolecular organizationthe LHCII and p-LHCII monolayers are composed of trimers assembled in either loosely packed homogeneous well-ordered monolayer areas or tightly packed heterogeneous disordered phase, with these two being in different proportions in the two types of monolayers.25 To characterize the LHCII conformation in monolayers at surface pressures below and above the transition surface pressure in the isotherms, i.e., at 10 and 30 mN/m, respectively, we apply PM-IRRAS; typical spectra are presented in Figure 1b,c. Structure and Orientation of LHCII at the Air/Buffer Interface at Low Surface Pressure. Figure 1b illustrates the amide I and II regions in the PM-IRRAS spectra of LHCII and p-LHCII monolayers at 10 mN/m. At this surface pressure both films are stable; relaxation processes do take place but are rather slow and do not affect the monolayer during spectra recording.25 The spectrum of the partly deprotonated complex (Figure 1b) displays only positive bandsamide I centered at 1648 cm−1 and amide II at 1534 cm−1; CH2 bending mode appears at 1447 cm−1 and CO ester carbonyl band (originating from lipids and chlorophylls) at 1730 cm−1. The positions and intensities of these bands clearly indicate that the protein secondary structure in the LHCII monolayer is predominantly composed of α-helices, as observed in solution.9,26,27 For comparison, Taneva et al.26 recorded spectra of loosely stacked and tightly stacked LHCII-lamellar aggregates in H2O and reported α-helix bands centered at 1654 and 1651 cm−1, correspondingly. The asymmetric shape of the amide I band (Figure 1b) and the shoulders at 1630 and 1615 cm−1 indicate the presence of a certain amount of parallel β-sheets and aggregated structures. LHCII forms stable trimers28 that selforganize into aggregates both in vivo and in vitro,29 so that the shoulder, formally assigned to β-sheets, actually arises from the association of neighboring trimers and is due to formation of hydrogen bonds between the carbonyl and amino groups of the α-helices of adjacent trimers.9 Furthermore, the shape of the amide I band in the PM-IRRAS spectrum is very similar to the FTIR spectrum of Langmuir−Blodgett (LB) monomolecular layer of LHCII, deposited on ZnSe monocrystal,30 and of liquid LHCII samples deposited on ZnSe crystal by partial evaporation.9,27 This strongly indicates that the protein retains its native conformation when transferred as a LB monolayer. The shape of the amide I band of proteins allows not only determination of the protein secondary structure but also evaluation of the molecular organization and orientation of the protein components.31,32 To qualitatively assign the individual secondary structure elements, decomposition and subsequent band fitting of the spectrum was performed. The Gaussian fitting of the amide I spectral region between 1700 and 1570 cm−1 yields five components (Figure 2a), quite similar in shape and frequencies to the components in the spectrum of LHCII LB monolayer deposited on ZnSe monocrystal.30 The most C
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Langmuir to the lumenal surface of the membrane,4−6 i.e., the mean tilt angle of the three main helices yields 50°. Figure 1b shows that the protonation (p-LHCII sample and a buffer subphase with pH 5.2) has a dramatic effect on the protein spectrum in monolayer at 10 mN/m. An entirely negative amide I band is observed, characterized by asymmetric and complex shape comprising two partly overlapping peaks and three shoulders. Because the decomposition of negative IR bands is very hazardous and might lead to a misleading interpretation, decomposition components of this band are not presented. The most intense peak is centered at 1675 cm−1, perfectly matching the position of the negative amide I band of α-helices with close-to-vertical orientation (tilt angle between 60 and 90°), predicted from spectra simulations, that always appears shifted to higher frequency.32 The IAI/IAII ratio for the 1530 cm−1 band is IAI/IAII = −1 ± 0.2, indicating almost perpendicular orientation of the α-helices with respect to the interface, i.e., θ close to 90°. Therefore, in p-LHCII monolayer the transition dipole moment of the amide II band lies in the plane of the helix, i.e., perpendicular to the buffer surface, whereas the transition dipole moment of the amide I band lies out of the plane of the helix, i.e., along the interface. Similarly to the partly deprotonated LHCII, the amide I band of p-LHCII consists of five components and the secondary structure elements of the complex are the sameα-helices (peak at 1675 cm−1), β-like structures (peak at 1637 cm−1 and shoulder at 1656 cm−1), turns (shoulder at 1696 cm−1), and aggregated strands (shoulder at 1618 cm−1) (Figure 2b). The pronounced red-shift of the bands from 1626 to 1637 cm−1 and from 1605 to 1618 cm−1 for LHCII and p-LHCII, respectively, suggests a marked weakening of the H-bonds between the trimers, as well as between the monomers comprising the trimers. To this point, we can conclude that the protonation of LHCII does not affect its secondary structure but induces conformational changes manifested by modification of the orientation of the α-helices and weakening of the interactions between and within the trimeric LHCII pigment−protein complexes. These results are in line with the considerable difference between the π/A isotherms of the LHCII and pLHCII monolayers at low surface pressures (Figure 1a).25 The mean area of one p-LHCII monomer in monolayer at 10 mN/m is by 1625 Å2 lower than that of one LHCII monomer (3565 versus 5190 Å2, Figure 1a) and is only slightly higher than the specific area, 3333 Å2, found for LHCII monomer in a trimer approximated to 100 × 100 Å rectangle.30 If one takes into account the projected area of an inclined cylinder-shaped monomer (Aprojected = 2rh sin θ + πr2 cos θ, where r is the radius and h is the height of the cylinder), the tilt angle θ of the cylinder can be calculated. The structural model for LHCII obtained by electron diffraction of two-dimensional crystals5 reveals that the cylindrically shaped LHCII trimer has a transmembrane height of 60 Å. Using the mean surface area of the p-LHCII monomer at 10 mN/m (3565 Å2) and considering the fact that p-LHCII helices are almost vertical, we determined that r = 33.7 Å. On the basis of the aforementioned values, we calculated that the projected surface area of one LHCII monomer will be 5190 Å2, i.e., the mean monomer area of LHCII found from the π/A isotherms, only when LHCII helices are inclined at θ = 33°. The calculated tilt angle is comparable to the average angle ∼40° that we already predicted on the basis of the PM-IRRAS spectrum and almost matches the angle of 30° from crystallographic data.4−6 This provides
strong evidence that the protonation-induced change in the orientation of the LHCII helices at the interface is the main reason for the difference between the π/A isotherms of LHCII and p-LHCII monolayers found at low surface pressure. Structure and Orientation of LHCII at the Air/Buffer Interface at High Surface Pressure. Compression to 30 mN/m has a destabilizing effect on both LHCII and p-LHCII monolayers, and continuous compression is needed to keep the surface pressure constant during the spectra measurement. We have previously found that the protonation results in stabilization of the LHCII monolayer, due to the decreased electrostatic repulsion and increased hydrophobicity of the pLHCII complexes.25 Raising the surface pressure from 10 to 30 mN/m exerts considerable influence on the overall shape of the spectra. The PM-IRRAS spectra of LHCII and p-LHCII monolayers are very similar and show almost identical features (Figure 1c). The amide I band splits to one negative component at 1698 cm−1 for LHCII/1702 cm −1 for p-LHCII and one positive component at 1649 cm−1 for LHCII/1656 cm−1 for p-LHCII. The negative bands appear at wavenumbers too high to be ascribed to vertical orientation of the helices. They might rather be associated with the δ(OH2) deformation mode of liquid water arising from anomalous dispersion of the real part of the water refractive index, appearing in the PM-IRRAS spectra as a broad band at ∼1700 cm−1.33,34 The bands originating from aggregated β-like structures are located at 1627 cm−1/1639 cm−1, and those at 1664 cm−1/1671 cm−1 are assigned to βturns. The red-shift of all p-LHCII peak positions is indicative for protonation-induced reorganization of the pigment−protein complex. During LHCII compression the AI/AII ratio changes from 1.4 ± 0.2 at 10 mN/m to 1.0 ± 0.2 at 30 mN/m. Thus, we estimate a mean tilt angle θ ≈ 45 ± 2° for both LHCII and p-LHCII helices at 30 mN/m. On the basis of the spectral data we consider that, along with the higher surface packing density of the complexes, conformational and orientational transitions occur during the compression of LHCII monolayers. Recent 77 K infrared spectra of dark-adapted and illuminated LHCII lamellar fragments, deposited on ZnSe crystal, demonstrated a red-shift of the C−OH stretching band, due to excitation quenching.35 The light-induced processes affecting LHCII-bound xanthophylls are associated with breaking of hydrogen bonds, in which C−OH groups are involved. In Figure 3a we present the spectra of LHCII and p-LHCII monolayers (at 30 mN/m) in the region 1090−1000 cm−1, where C−O stretching vibrations are observed.36 For comparison, both spectra are normalized, so that they have the same integrated area in the presented region. The spectral shift toward higher wavenumbers reflects weakening of the Hbonding between xanthophylls and the protein scaffold of pLHCII due to protonation. This shift is more clearly expressed in Figure 3b, where the difference between p-LHCII and LHCII spectra shows a maximum in the high-wavenumbers and a minimum in the low-wavenumbers region. Therefore, an analogous spectral shift is induced by LHCII protonation (this work) and LHCII illumination.35 Because illumination causes thinning and increase in the hydrophobicity of the thylakoid membrane,37 it can be expected that the protonation of LHCII would also increase the hydrophobicity of the complex itself and respectively of the LB films, built by transferring LHCII and p-LHCII monolayers on glass slides at 10 and 30 mN/m. Therefore, the surfaces of D
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when compressed to 10 mN/m both surfaces are hydrophilic (partly deprotonated LHCII being more hydrophilic than pLHCII) but at 30 mN/m comparable hydrophobicities for LHCII and p-LHCII approaching the boundary between the hydrophilic and hydrophobic materials are observed. The data in Table 1 illustrate that the protonation of LHCII complex has an unambiguous effect on the surface hydrophobicity of LB films transferred at 10 mN/m and has no impact on the films transferred at 30 mN/m. Although the intermolecular interactions for LHCII and p-LHCII in the monolayers at 30 mN/m differ, the orientation of the helices at the interface is identical θ ≈ 45°. These latter similarities are fully in line with the close resemblance of the π/A isotherms at high surface pressure and confirm our finding that the mean monomer area of the pigment−protein complex at the interface is mainly determined by the orientation of α-helices with respect to the monolayer plane. On the basis of the obtained data, we propose a model of the changes in the orientation of the three hydrophobic transmembrane helicesA, B, and C in the LHCII and p-LHCII complexes occurring upon protonation and increase of surface pressure (Figure 5).
Figure 3. (a) PM-IRRAS spectra of LHCII (blue line) and p-LHCII (red line) monolayers, at surface pressure 30 mN/m, in the spectral region of C−OH stretching vibrations. (b) Difference PM-IRRAS spectrum (p-LHCII − LHCII), representing the effect of protonation.
the LHCII LB films were characterized by the contact angle of a water drop that yielded Θ < 60 for 10 mN/m and Θ > 65 for 30 mN/m (Figure 4, Table 1). According to Vogler,38
Figure 5. Sketch of the change of the LHCII α-helices orientation as a result of protonation and monolayers compression, displaying only the main three helical residues for clarity. Figure 4. Shape of water drops formed on the LHCII and p-LHCII surfaces at surface pressures 10 and 30 mN/m.
Considering that the membrane lateral pressure was estimated to be between 25 and 34 mN/m,39−41 our results at a surface pressure of 30 mN/m are consistent with the recent finding that the photoprotective switch of LHCII does not affect the tilting of the transmembrane α-helices A and B but does affect the arrangement of the helical segments and loops close to the interface.42
Table 1. Average Water Contact Angle (Θ) of LB Films Determined for LHCII and p-LHCII Transferred at 10 and 30 mN/m (± Standard Deviation) Θ (degree) surface pressure
LHCII
p-LHCII
10 mN/m 30 mN/m
51.8 (±1.7) 67.1 (±2.0)
59.2 (±2.5) 65.9 (±2.0)
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CONCLUSIONS In this work we report that the native α-helical structure of LHCII is retained in monolayers and that LHCII protonation triggers strong changes in the conformation of the protein, in the orientation of the helices (from inclined (θ ≈ 40°) to nearly vertical (θ ≈ 90°)), and in the hydrophobicity of LHCII monolayers at surface pressure 10 mN/m. At surface pressure
biomaterials with contact angles greater than 65° are classified as hydrophobic while surfaces with contact angles less than 65° are classified as hydrophilic. Therefore, we can conclude that E
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HgCdTe detector. To ensure good signal/noise ratio, 800 interferograms, representing an acquisition time of 10 min, were coadded. The principle of the method and its application to Langmuir films at the air/water interfaces, as well as PMIRRAS experiments, were described previously.32,33 The PMIRRAS spectra of the samples were normalized by the subphase spectra. The monolayers were built as described above, compressed to 10 or 30 mN/m surface pressure, and kept constant during the spectra acquisition. The monolayers were left to equilibrate for 1 min before the spectral data collection. The spectra with positive amide I bands were decomposed by means of Gaussian fitting in amide I regions; the correlation factor for each peak was no less than 0.999. Prior to the decomposition the exact band positions were determined from the second derivative spectra. The spectra were analyzed based on the PM-IRRAS “surface-selection rule” and simulated spectra of proteins in the spectral region of the amide I and amide II bands at different orientations of α-helices and βsheets.31,32 Static Contact-Angle Measurements. The hydrophilic/ hydrophobic surface properties of the LB films were assessed by a contact-angle goniometer (Ramé-Hart Automated Goniometer model 290) equipped with a video-capturing system. Static contact angles were measured by the sessile drop method.47 For the contact-angle measurements 3 μL of ultrapure water were placed on the leveled surface of the sample by microsyringe, forming a single sessile drop. The left and right angles were measured at 4−5 different spots for each specimen.
typical for biological membranes (30 mN/m), the protonation does not affect the secondary structure and orientation of LHCII transmembrane α-helices but weakens the interaction between the monomers within a trimer as well as the trimer− trimer interactions. Such structural flexibility as a result of the LHCII conformational switch is probably needed for its photoprotective function. Providing that these processes occur also in the native thylakoid membrane, they might have consequences with important physiological significance facilitated lateral diffusion of the trimers (shuffling of LHCII between the two photosystems, detachment from the PSII supercomplex, and LHCII-enriched domains formation) and enhanced accessibility for the enzyme violaxanthin de-epoxidase (which converts the LHCII-bound violaxanthin to zeaxanthin).
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EXPERIMENTAL SECTION Isolation of LHCII. LHCII lamellar macroaggregates were isolated from pea thylakoid membranes according to the protocol of Krupa et al.,43 modified by Simidjiev et al.;44 0.7% Triton X-100 (Sigma-Aldrich) was used for solubilization of thylakoid membranes, resulting in loosely stacked two-dimensional lamellae highly enriched in LHCII. The chlorophyll concentration was determined spectrophotometrically.45 The chlorophyll a/b ratio of the LHCII preparations was 1.16 ± 0.04. Partly deprotonated and protonated LHCII samples were prepared by washing the LHCII suspension three times in 50 mM Tricine buffer with pH 7.8 or 5.2, respectively. Three independent LHCII preparations were investigated in this work. Monolayers Formation. All experiments were carried out at identical experimental conditions using a Langmuir balance with Teflon trough, Wilhelmy dynamometric system, and LB equipment for LB films deposition. Twenty-five μL of 40 μM LHCII suspension were spread on a buffer subphase containing 50 mM Tricine and 500 mM NaCl, at pH 7.8 or 5.2 (water from Purelab Option-Q purification system with specific resistance of 18 MΩ/cm), providing a sufficiently high initial monomer area of 8300 Å2. After 30 min equilibration, the Langmuir monolayers were compressed at a rate of 900 Å2/ monomer·min to the desired surface pressure, which was automatically maintained during the PM-IRRAS measurements and LB films deposition. The presence of 500 mM NaCl in the subphase is essential to avoid the sinking of the protonated LHCII complexes. In our previous study25 we proved that extrusion of the protonated LHCII from the water surface takes place during monolayer compression, but increase in the subphase salt concentration eliminates this effect. Therefore, in the present work we use a buffer subphase containing 50 mM Tricine and 500 mM NaCl. Regardless of the subphase salt concentration, submerging of the partly deprotonated LHCII does not take place.25 The addition of monovalent cations to the subphase, in high concentration, is also known to induce destacking of thylakoid membranes46 and thus prevents the natural face-to-face aggregation observed in granas. LHCII and p-LHCII monolayers were transferred from the buffer subphase to round glass coverslips (diameter of 12 mm) at surface pressures of 10 and 30 mN/m. At least five independent measurements were performed for each of the three individual LHCII preparations and for each experimental condition. PM-IRRAS Measurements. PM-IRRAS spectra of the LHCII monolayers were recorded at 8 cm−1 resolution on a Nicolet 740 spectrometer equipped with liquid nitrogen-cooled
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AUTHOR INFORMATION
Corresponding Author
*
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Ministry of Education and Science of Bulgaria, project BG051PO001-3.3.05-0001 “Science and Business” (Grant no. DO2-846/10.10.13). The authors are grateful to Dr. M. Busheva and Dr. S. Stoichev for LHCII preparations.
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REFERENCES
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DOI: 10.1021/acs.langmuir.5b02653 Langmuir XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.langmuir.5b02653 Langmuir XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.langmuir.5b02653 Langmuir XXXX, XXX, XXX−XXX
