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Investigation on the Thermodynamic Dissociation Kinetics of Photosystem II Supercomplexes to Determine the Binding Strengths of Light-Harvesting Complexes Eunchul Kim, Ryutaro Tokutsu, and Jun Minagawa J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b12417 • Publication Date (Web): 18 Jan 2018 Downloaded from http://pubs.acs.org on January 18, 2018

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The Journal of Physical Chemistry

Investigation on the Thermodynamic Dissociation Kinetics of Photosystem II Supercomplexes to Determine the Binding Strengths of Light-Harvesting Complexes Eunchul Kim1, Ryutaro Tokutsu1,2,3, and Jun Minagawa1,2,3* 1

2

Division of Environmental Photobiology, National Institute for Basic Biology, Okazaki 444-8585, Japan

Department of Basic Biology, School of Life Science, Graduate University for Advanced Studies, Okazaki

444-8585, Japan. 3

Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Saitama

332-0012, Japan.

Corresponding Author *E-mail: [email protected]

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ABSTRACT The photosystem II (PSII) supercomplex splits water utilizing light energy, and is composed of a core dimer-complex surrounded by light-harvesting complexes (LHCs). In green algae, the major LHCs which are LHCII trimers, have thus far been categorized into strongly, moderately, or loosely binding LHCII trimers based on their predicted binding to core complexes. However, the binding energies have been indirectly predicted based on the presence or absence of LHCII trimers in the PSII supercomplex under electron microscopy, and have not been determined experimentally. In this study, we investigated the binding of LHCII trimers by analyzing thermodynamic dissociation kinetics using isolated PSII supercomplexes. We identified two activation energies for dissociation of LHCII trimers, 54 ± 19 kJ/mol and 134 ± 8 kJ/mol. This result indicated the types of intermolecular interactions between LHCII trimers and core complexes.

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INTRODUCTION One of the most important light energy conversion processes, photosynthesis, takes place in the thylakoid membranes of photosynthetic organisms. Photosystem I and II complexes (PSI and PSII) absorb light and generate electron flow and proton gradient across thylakoid membranes in the chloroplast for NADPH and ATP synthesis1. To allow efficient light-harvesting, PSI and PSII are associated with corresponding light-harvesting complex (LHC) proteins and form individual supercomplexes. In particular, photosystem complexes in the green alga Chlamydomonas reinhardtii have been demonstrated to have more LHCs than higher plants. The PSI supercomplex is composed of a core monomer and nine distinct LHCI monomers in C. reinhardtii2-3. The PSII supercomplex is composed of a core dimer and is surrounded by four minor LHC monomers and six major LHC trimers (LHCII trimers)4-5. The LHCII trimers have been categorized as strongly (S), moderately (M), and loosely (L) bound trimers (a complete PSII particle is designated as C2S2M2L2, where ‘C’ is a core complex)4-5. Thus far, binding strength has been indirectly assessed based on probability of LHCII trimer occurrence in PSII supercomplexes examined using electron microscopy6. Therefore, variations in binding strength of the LHCII trimers remain to be investigated, and investigation of the binding energies would also provide insight into the structure of the PSII supercomplex. The challenge in this context has been the lack of suitable methods to determine internal binding properties of membrane protein complexes, although various methods have been developed to study interactions between soluble proteins7. Here, we present a new approach to determine the binding strength of a membrane protein by analyzing thermodynamic dissociation kinetics. We examined the binding strength of LHCII trimers in a PSII supercomplex isolated from the green alga C. reinhardtii, using steady-state and time-resolved fluorescence spectroscopy. 3



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MATERIALS AND METHODS Isolation of photosystem II supercomplexes Thylakoid membranes were first isolated from wild-type (137c) C. reinhardtii cells grown under light with 20 µmol photons m-2 s-1 with 5 % CO2 bubbling and stirring. Then, the thylakoid membranes were solubilized with dodecyl-α-maltoside (α-DM; Affymetrix) in 25 mM MES buffer (pH 6.5) and 1 M betaine, and PSII supercomplexes were purified using sucrose density gradient ultracentrifugation as described by Tokutsu et al.4, 8.

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) The PSII-LHCII supercomplexes were immediately mixed with 4 x NativePAGETM sample buffer and Coomassie Brilliant Blue G-250 at a final concentration of 0.5 % and incubated on ice for 1 min. The samples were then separated on 4–16 % Bis-Tris NativePAGETM gel following the manufacturer’s instructions (Thermo Fisher Scientific).

Steady-state 77 K fluorescence spectroscopy Steady-state fluorescence emission spectra were obtained at 77 K using a spectrofluorometer, FluoroMax (HORIBA Jobin-Yvon). Chlorophyll was excited at 440 nm (slit width = 2 nm) and the emission detected in the 620–800 nm range at 1 nm-intervals with a 1 nmslit width. Sample concentration was set to 1 µg chlorophyll per ml (optical density at the absorption peak at 680 nm was lesser than 0.1) to avoid reabsorption of emission from the sample. Samples were frozen in a glass tube using liquid nitrogen.

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Time-resolved fluorescence spectroscopy Time-resolved fluorescence decay was obtained using a time-correlated single photon counting system, FluoroCube (HORIBA Jobin-Yvon). A picosecond pulse diode laser (N-440L, HORIBA Jobin-Yvon) was used to excite chlorophyll at 441 nm with a 1 MHz repetition rate (1.02 pJ per a pulse). Fluorescence emission was detected at 685 nm with a large slit width (FWHM = 32 nm) to cover overall fluorescence from chlorophyll. Deconvolution of fluorescence decay curves was performed using IgorPro 7 (WaveMetrics) with a triple-exponent function and an instrument-response function of the system (full-width half maximum = 200 ps). We used only the fitting results with reduced 𝝌2 values of 0.9–1.2 with traces of 4 ns-fluorescence lifetime component amplitudes. Temperature was controlled using a temperature water bath and precise temperature was measured using a thermometer.

RESULTS AND DISCUSSION PSII supercomplexes were solubilized with dodecyl-α-maltoside (α-DM; Affymetrix) in 25 mM MES buffer (pH 6.5) and purified using sucrose density gradient ultracentrifugation (Figure 1A), as described in previous reports4, 8. The isolated PSII supercomplexes showed a chlorophyll a/b ratio of 2.03, which corresponds to the chlorophyll a/b ratio of C2S2M2L1-2 PSII supercomplexes (Figure S1 and Tables S1 and S2). The isolated PSII supercomplexes were stored at 38 °C for 0, 0.5, 1, 3, or 7 hours and resolved via blue-native PAGE (BN-PAGE) to monitor physical dissociation of the PSII supercomplexes (Figure 1B). Heat treatment caused a decrease in size of the PSII-LHCII supercomplexes, and the amount of free LHCII trimers increased compared to that before heat treatment (time zero), clearly demonstrating physical dissociation of LHCII trimers from the PSII core. We designated each band based on previous reports9-10. This 5



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Figure 1. Dissociation of LHCII trimers from PSII supercomplexes. (A) Result of sucrose density gradient (SDG) ultracentrifugation of solubilized thylakoid membranes. (B) Blue native PAGE of PSII supercomplexes (90 µg of chlorophylls / mL) stored at 38 °C for 0, 0.5, 1, 3, or 7 hours, and isolated LHCII trimers. (C) 77 K fluorescence emission spectra. (D) fluorescence decay of the samples in (B) after dilution to a concentration of 2 µg chlorophylls / mL (OD at 680 nm ≈ 0.1). Spectra were normalized at 684 nm. Fluorescence decay measurements were obtained with a wide wavelength range of emission (685 nm ± 16 nm).

physical dissociation of LHCII trimers was accompanied by specific changes in fluorescence emission spectra and decays. In the case of intact PSII-LHCII supercomplexes, the majority of 6


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fluorescence at 77 K is emitted from core complexes due to efficient energy transfer from LHCII trimers to the core complexes. However, when LHCII trimers are dissociated from the PSII supercomplex, the proportion of fluorescence from LHCII trimers increases because excitation energy present in the trimers cannot be transferred to the core. In this regard, 77 K fluorescence emission spectra exhibit a change in the ratio of fluorescence intensity between 678 nm and 684 nm (Figure 1C), where fluorescence peaks originate from LHCII trimers and PSII core complexes, respectively11-13. In time-resolved fluorescence decay, we found that the average fluorescence lifetime was extended in dissociated complexes due to increase in the amplitude of a 4 ns-lifetime component (Figure 1D). This 4-ns component was assigned to free LHCII on the basis of identical fluorescence lifetime and spectrum. To monitor dissociation kinetics of the LHCII trimers quantitatively, we used the amplitude of the 4 ns-component as a signature of dissociated LHCII trimers (Figure 2). Because a 440-nm laser excites chlorophyll a and chlorophyll b almost equally, amplitude of the 4 ns-component reflects the ratio between chlorophyll bound to dissociated LHCII trimers and total chlorophyll bound to proteins, irrespective of chlorophyll a/b ratio. Moreover, fluorescence lifetime (~ 4 ns) of the free LHCII trimers was clearly distinct from that (0.2 and 1.4 ns) of the core complexes (Fitting results are shown in Figure S2 and Table S3)11, 13-14, where spectral features allowed us to separate fluorescence components. Time-dependent changes in the amplitude of the 4 nscomponent showed bi-phasic curves (Figure 2). The initial phase (0–20 min) reflects a faster dissociation of LHCII trimers than the late phase (20–1800 min). Accordingly, to fit curves obtained at temperatures lower than 23 °C, a bi-exponential function yielded a better fitting result than a single-exponential function, indicating more than two different binding modes between LHCII trimers and the PSII-LHCII supercomplex. 7



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Figure 2. Bi-phasic trace of the 4 ns-fluorescence lifetime component amplitudes at 18 °C. Biexponential and single-exponential functions were applied to fit the bi-phasic trace. The inset is the magnified plot showing the fitting qualities of bi-exponential and single-exponential fitting lines.

To determine binding strength of the LHCII trimers, we obtained traces of dissociation kinetics at various temperatures (Figure 3A and Figure S3). The traces of dissociation kinetics were

apparently

dependent

on

temperature,

suggesting

that

the

dissociation

was

thermodynamically regulated. When temperature increased, dissociation of LHCII trimers was dramatically accelerated. Consistently, the two respective rate constants derived via bi-exponential fitting gave good linear relationships in the Arrhenius plot of logarithm of rate constant (ln k) versus inverse temperature (1/T) (Figure 3B). As a result, the two activation energies for dissociation of the LHCII trimers were determined based on the Arrhenius equation to be 54 ± 19 kJ/mol and 134 ± 8 kJ/mol. In this study, we obtained two rate constants of dissociation kinetics from isolated PSII supercomplexes, although three types of LHCII trimers were predicted based on previous reports48


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Figure 3. Temperature dependence of dissociation kinetics and the Arrhenius plot. (A) Traces of the 4 ns-fluorescence lifetime component amplitudes at 12, 23, 32, 38, and 45 °C. Gray lines represent fitting lines of a bi-exponent function. (B) Arrhenius plot of dissociation rate components and its linear fitting lines. Error ranges represent standard deviations.

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. Relative amplitudes of the two obtained rate constants were ~ 4 ± 2 % for 54 ± 19 kJ/mol and ~

72 ± 2 % for 134 ± 8 kJ/mol. The remaining ~ 26 ± 3 % of LHCII trimers appeared to be already dissociated during the process of dilution for fluorescence measurements, based on two facts: (i) The BN-PAGE with concentrated samples (90 µg of chlorophyll / mL) before heat-treatment did not show any free LHCII trimers (0 h in Figure 1B), which would indicate that most LHCII trimers 9



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in the samples were associated with PSII supercomplexes (ii) The amplitude of the 4 ns-component in fluorescence decay measurements obtained from diluted samples before heat-treatment was ~ 26 ± 3% compared with the amplitude at fully dissociated conditions. The amplitude of the 4 nscomponent was ~ 62 ± 5 % at equilibrium in diluted samples (2 µg of chlorophylls / mL), which was consistent with the ratio of chlorophylls belonging to LHCII trimers in the C2S2M2L2 PSII supercomplex (Figure S4 and Table S2). Taken together, our results show that 26 ± 3 % of LHCII trimers can be expected to have similar or much weaker activation energy of dissociation than 54 ± 19 kJ/mol, due to dissociation during dilution.

CONCLUSIONS We investigated thermodynamic dissociation kinetics of a PSII supercomplex using fluorescence spectroscopy, and found that LHCII trimers in the PSII supercomplex of C. reinhardtii possess at least two binding strength values. We expect that this approach for investigating thermodynamic dissociation kinetics is applicable to other membrane protein complexes in studies of membrane protein binding properties.

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ASSOCIATED CONTENT Supporting Information Chlorophyll a/b ratio of an isolated PSII supercomplex; examples of decay fitting results; concentration-dependence of fluorescence decays; Tables about the number of chlorophylls and chlorophyll a/b ratio of photosystem II supercomplexes (PDF)

ACKNOWLEDGMENT This work was supported by Grant-in-Aid from Japan Society for the Promotion of Science (JSPS) KAKENHI (JP16H06553, JP26251033) and from International Research Fellowship of JSPS (to E.K.). We thank Dr. R. N. Burton Smith for critical reading of the manuscript.

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REFERENCES (1) Blankenship, R. E. Molecular Mechanisms of Photosynthesis; Blackwell Science: Oxford, Malden, MA, 2002. (2) Stauber, E. J.; Fink, A.; Markert, C.; Kruse, O.; Johanningmeier, U.; Hippler, M. Proteomics of Chlamydomonas reinhardtii Light-Harvesting Proteins. Eukaryot. Cell 2003, 2, 978-994. (3) Drop, B.; Yadav, K. N. S.; Boekema, E. J.; Croce, R. Consequences of State Transitions on the Structural and Functional Organization of Photosystem I in the Green Alga Chlamydomonas reinhardtii. Plant J. 2014, 78, 181-191. (4) Tokutsu, R.; Kato, N.; Bui, K. H.; Ishikawa, T.; Minagawa, J. Revisiting the Supramolecular Organization of Photosystem II in Chlamydomonas reinhardtii. J. Biol. Chem. 2012, 287, 3157431581. (5) Drop, B.; Webber-Birungi, M.; Yadav, S. K.; Filipowicz-Szymanska, A.; Fusetti, F.; Boekema, E. J.; Croce, R. Light-Harvesting Complex II (LHCII) and Its Supramolecular Organization in Chlamydomonas reinhardtii. Biochim. Biophys. Acta 2014, 1837, 63-72. (6) Boekema, E. J.; van Roon, H.; Dekker, J. P. Specific Association of Photosystem II and LightHarvesting Complex II in Partially Solubilized Photosystem II Membranes. FEBS Lett. 1998, 424, 95-99. (7) Kastritis, P. L.; Bonvin, A. M. On the Binding Affinity of Macromolecular Interactions: Daring to Ask Why Proteins Interact. J. R. Soc., Interface 2013, 10, 20120835. (8) Tokutsu, R.; Minagawa, J. Energy-Dissipative Supercomplex of Photosystem II Associated with LHCSR3 in Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 1001610021.

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