^^^^^^^^^^^^ ЭКСПЕРИМЕНТАЛЬНЫЕ ^^^^^^^^^^^^
СТАТЬИ
581.1
THERMAL STABILITY STUDIES OF PHOTOSYSTEM II COMPLEXES RECONSTITUTED INTO PHOSPHATIDYLCHOLINE LIPOSOMES1
© 2014 F. Zhou*, N. W. Qiu**, Z. J. Gu*, B. J. Zhang*, C. Hua*
*School of Biochemical and Environmental Engineering, Nanjing Xiaozhuang University, Nanjing, P.R. China **College of Life Science, Qufu Normal University, Qufu, P.R. China Received October 15, 2012
Light-harvesting II complexes (LHCII) and photosystem II core complexes (PSIICC) were isolated from spinach (Spinacia oleracea L.) and reconstituted into phosphatidylcholine liposomes and, under heat stress, PSIICC—LHCII proteoliposomes were found to exhibit significantly higher oxygen evolution activity than PSIICC proteoliposomes lacking LHCII. In the presence of LHCII, the temperature of a 10-min heat stress that caused semi-inactivation of oxygen-evolving activity in these liposomes increased from 34 to ~37°C and the total inactivation temperature increased from ~50 to ~60°C. Moreover, with heat stress, decreases in the absorbance and fluorescence spectra of PSIICC—LHCII proteoliposomes were smaller than in LHCII-lacking PSIICC proteoliposomes. These results demonstrated that reconstitution of PSII into liposomes with LHCII increased the antenna size and light harvesting cross-section of PSII and thus, under heat stress, enhanced PSII photochemical activity and thermal stability.
Keywords: Spinacia oleracea — light-harvesting complex II—proteoliposomes — heat stress — O2 evolution activity
ФИЗИОЛОГИЯ РАСТЕНИЙ, 2014, том 61, № 1, с. 31-37
УДК
DOI: 10.7868/S0015330314010199
INTRODUCTION
Light absorption and energy transfer in photosynthesis take place in thylakoid membranes that contain pigment—protein complexes embedded in a lipid matrix. Photosystem II (PSII) is one of the integral multi subunit pigments—protein complexes in thylakoid membranes, which uses solar energy to split water molecules into free protons and molecular oxygen. The PSII "supercomplex" can be split into two parts: an outer antenna portion or light-harvesting complex (LHCII) and a PSII core complex (PSIICC) [1]. Plants have developed a very complicated light-harvesting system in the PSIICC—LHCII supercomplex, which enables efficient energy absorption and transfer to the reaction center where the primary photochemical reaction occurs [2]. The affinity of LHCII for PSIICC is an important factor for controlling the energy balance in PSII supercomplexes. It has been observed that the stoichiometry of LHCII/PSIICC in
1 This text was submitted by the authors in English.
Abbreviations'. BBY — PSII-enriched membranes; Chl — chlorophyll; LHCII — light-harvesting complexes of PSII; PC — phosphatidylcholine; PSII — photosystem II; PSIICC — photosystem II core complexes..
Corresponding author. Feng Zhou. School of Biochemical and Environmental Engineering, Nanjing Xiaozhuang University, Nanjing, Jiangsu 211171, P.R. China. Fax. +0086-25-8617-8262; e-mail. zfibcas@163.com
the PSII supercomplexes is changed not only during different thylakoid developmental stages but also under different energy excitation conditions [3, 4]. Moreover, the lipid—protein interaction is a key factor mediating the structural and functional interactions among the subunits of these supercomplexes [5, 6].
It is difficult to interpret the functions of the membrane proteins in their native environment due to restrictions arising from the complexity of the native membranes and interference from other membrane constituents or reactions [7]. Thus, in liposome research lipid vesicles incorporating purified membrane proteins have been a powerful tool for elucidating both the functional and structural aspects of these membrane-associated proteins [8]. In the present study, the goal was to illuminate the interactions between PSIICC and LHCII under heat stress. The native thylakoid membrane environment was modulated by incorporating isolated LHCII and PSIICC into liposome membranes composed of phosphatidylcholine (PC) and then studying the proteoliposomes in terms of the electron transport activity of PSII, absorption spectrum, and fluorescence emission spectrum of PSIICC or LHCII—PSIICC proteoliposomes under different temperature regimes. These results revealed that, under heat stress, PSII photochemical activity and thermal stability were enhanced when LHCII was included in the reconstituted proteoliposomes.
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MATERIALS AND METHODS
Preparation of PSIICC complexes. PSII-enriched membranes (BBY) were isolated from spinach (Spina-cia oleracea L.) purchased from a local market, as described by Berthold et al. [9]. PSIICC was then purified from the BBY preparation according to a procedure by Ghanotakis et al. [10]. Briefly, BBY was resuspended in a buffer containing 0.4 M sucrose, 50 mM Mes-NaOH (pH 6.0), and 10 mM NaCl to a concentration of 2.5 mg chlorophyll (Chl)/mL, and mixed with an equal volume of a solution of1.0 M sucrose, 50 mM Mes-NaOH (pH 6.0), 0.8 M NaCl, 10 mM CaCl2, and 70 mM n-octyl-p-D-glucopyra-noside. After incubation in darkness at 4°C for 10 min, the suspension was mixed with twice its volume of a buffer with 1.0 M sucrose, 50 mM Mes-NaOH (pH 6.0), 0.4 M NaCl, and 5 mM CaCl2 and incubated again for 5 min, followed by centrifugation at 40 000 g for 90 min. The supernatant was collected and desalted, the sucrose was removed by dialysis against 50 mM Mes-NaOH (pH 6.0), 10 mM NaCl, and 5 mM CaCl2, and the resulting solution was sedimented by centrifugation at 40000 g for 90 min. The pellet of PSIICC was resuspended in a solution containing 0.4 M sucrose, 50 mM Mes (pH 6.0), 10 mM NaCl, and 5 mM CaCl2, frozen in liquid nitrogen, and stored at -80°C.
Preparation of LHCII complexes. LHCII membrane fractions were prepared according to the method of Krupa et al. [11] with some modifications [12]. The BBY preparation was washed twice with a buffer of 5 mM EDTA, 50 mM sorbitol, and 50 mM Tricine-NaOH (pH 7.8), and centrifuged at 10000 g for 10 min. The pellet was resuspended in cold distilled H2O to 0.8 mg Chl/mL, Triton X-100 added to a final concentration of 0.7% (w/v), and the resulting suspension was incubated at room temperature with continuous stirring for 30 min, followed by centrifugation at 30000 g for 40 min. KCl and MgCl2 were added to the Triton X-100 supernatant to final concentrations of 100 and 20 mM, respectively, and the mixture was stirred gently for precipitation of crude LHCII. The suspension was then layered on 0.5 M sucrose (sucrose volume >3x of LHCII suspension volume) and centrifuged at 10000 g for 10 min. The resulting pellet was resuspended in 50 mM Tricine-NaOH buffer (pH 7.8) containing 100 mM sorbitol to ~0.8 mg Chl/mL. Triton X-100 (5% (w/v) stock solution) was then added to obtain a detergent/Chl ratio of 10/1 (w/w). After brief stirring, LHCII was precipitated with potassium and magnesium salts, as above, and the suspension was gently stirred to allow good LHCII precipitation, layered on 0.5 M sucrose solution, and centrifuged at 30000 g for 40 min. For the final purification, the pellet was suspended in 50 mM Tricine-NaOH buffer (pH 7.8) containing 100 mM sorbitol and precipitated with 1 M KCl and 1M MgCl2, as described above but lacking Triton X-100. The resulting
LHCII suspension was layered on 0.5 M sucrose solution, centrifuged at 10000 g for 10 min, resuspended in 50 mM Tricine-NaOH buffer (pH 7.8), frozen in liquid nitrogen, and stored at -80°C. Chl concentrations were determined in 80% aqueous acetone (v/v) solutions using the method of Arnon [13].
SDS-PAGE of Chl—protein complexes. The
polypeptide compositions of different Chl-protein complexes were evaluated by denaturing SDS-PAGE with 15% acrylamide, 6 M urea. The PAGE was run in the buffer system of Laemmli [14] and then was stained with Coomassie Brilliant Blue R-250.
Reconstitution of PSIICC and LHCII complexes into liposomes. PC was purchased from "Sigma" (P5638; United States; fatty acid contents: approximately 13% C16:0 (palmitic), 4% C18:0 (stearic), 10% C18:1(oleic), 64% C18:2 (linoleic), and 6% 18:3 (linolenic) with other fatty acids being minor contributors) and dissolved in chloroform, which was then slowly removed by rotary evaporation to produce a thin lipid film on the inner glass wall. The film was hydrated in a buffer of 10 mM Tris-HCl (pH 7.5) and 10 mM NaCl at 5 mg PC/mL, and liposomes were formed according to our method [15]. Suspensions of isolated PSII and LHCII were sonicated at 4°C in a sonicator for 5 min and these Chl-protein complexes reconstituted together or separately into liposomes by mixing with the dispersed lipid at a lipid/protein ratio of 4/1 (w/w), according to the methods of Larkum and Anderson [16], while the corresponding Chl/li-pid molar ratios were 1/20 and 1/13 (PSIICC and PSIICC-LHCII), respectively. The Chl/protein mol ratios of the chlorophyll-protein complexes were designed according to those of isolated complexes to emulate the native thylakoid membrane environment. The resulting mixture was immediately frozen in liquid nitrogen, incubated at -196°C for 1 min, thawed in water at 20°C, and finally sonicated at 4°C in a sonicator for 8 min. Heat treatment of PSIICC and PSIICC-LHCII proteoliposomes was performed by incubating them at high temperatures in the dark for 10 min.
Transmission electron microscopy (EM). A small droplet (10 ^L) of liposome suspension was applied to a holey carbon film on a 500 mesh copper grid. The specimens were observed under a JEM-100 CX transmission electron microscope after negative staining with 1% uranium acetate.
Absorption spectra measurements. Absorption spectra were recorded at room temperature with a Shi-madzu UV-Vis 2550 spectrophotometer ("Shimad-zu", Japan) using 1 cm-path length cuvettes. The incubation medium contained 10 mM Tris-HCl (pH 7.5) and NaCl.
Fluorescence measurements. Sample fluorescence spectra were obtained using a Hitachi F-2500
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