Oligomerization state and pigment binding strength of the peridinin-Chl a-protein

FEBS Letters 589 (2015) 2713–2719

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Oligomerization state and pigment binding strength of the peridinin-Chl a-protein Jing Jiang a, Hao Zhang b, Xun Lu c, Yue Lu b, Matthew J. Cuneo c, Hugh M. O’Neill c, Volker Urban c, Cynthia S. Lo a, Robert E. Blankenship b,d,⇑ a

Department of Energy, Environmental & Chemical Engineering, Washington University in St. Louis, St. Louis, MO 63130, USA Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130, USA Center for Structural Molecular Biology, Biology and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA d Department of Biology, Washington University in St. Louis, St. Louis, MO 63130, USA b c

a r t i c l e

i n f o

Article history: Received 31 May 2015 Revised 13 July 2015 Accepted 20 July 2015 Available online 1 August 2015 Edited by Richard Cogdell Keywords: Photosynthesis Light-harvesting Dinoflagellate Symbiodinium Chlorophyll Peridinin Native mass spectrometry

a b s t r a c t The peridinin-chlorophyll a-protein (PCP) is one of the major light harvesting complexes (LHCs) in photosynthetic dinoflagellates. We analyzed the oligomeric state of PCP isolated from the dinoflagellate Symbiodinium, which has received increasing attention in recent years because of its role in coral bleaching. Size-exclusion chromatography (SEC) and small angle neutron scattering (SANS) analysis indicated PCP exists as monomers. Native mass spectrometry (native MS) demonstrated two oligomeric states of PCP, with the monomeric PCP being dominant. The trimerization may not be necessary for PCP to function as a light-harvesting complex. Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction The peridinin-chlorophyll a-protein (PCP) is one of the major light harvesting complexes (LHCs) in photosynthetic dinoflagellates [1–3]. PCPs share no sequence similarity to other LHCs [4]. They are water-soluble and located in the luminal space of thylakoid membranes [3,5], serving as light harvesters together with the membrane-intrinsic LHCs (chlorophyll a-chlorophyll c2-peridinin-proteins, apcPCs) in the peridinin-containing dinoflagellates. Instead of chlorophylls, they have the blue-green (470–550 nm) absorbing carotenoid peridinins as primary

Author contributions: REB and CSL designed and supervised the project. JJ isolated the protein, carried out size exclusion chromatography experiments, and assisted in mass spectrometry experiments and neutron scattering experiments. XL, MJC and HMO’N carried out neutron scattering experiments. VU supervised neutron scattering experiments. HZ carried out mass spectrometry experiments. YL assisted in protein isolation and mass spectrometry experiments. JJ, CSL and REB wrote and revised the manuscript. ⇑ Corresponding author at: Washington University in St. Louis, One Brookings Drive, Campus Box 1137, St. Louis, MO 63130, USA. E-mail address: [email protected] (R.E. Blankenship).

pigments. PCPs of distinct lengths, pigment contents, amino acid sequences and spectroscopic properties were found in dinoflagellates. Among different PCPs, the structures of main form PCP (MFPCP) and high-salt PCP (HSPCP) from Amphidinium carterae have been resolved to 2.0 Å and 2.1 Å, respectively [3,6]. A higher resolution structure of 1.5 Å was recently achieved in recombinant PCP [7]. The X-ray crystal structure of MFPCP has revealed a trimer, in which each subunit folds in a twofold pseudosymmetry (a monomer has two pseudo-identical domains), holding 8 peridinins and 2 Chl a molecules. On the other hand, HSPCP, which contains 6 peridinins and 2 Chl a molecules, and recombinant PCP, which is one half of the MFPCP, crystallized as monomers. It is not clear if the trimerization is common for PCPs or the MFPCP trimer is an exception, although it has been suggested that the non-crystallographic trimer of MFPCP is relevant for the in vivo oligomeric state in the thylakoid lumen based on analytical ultracentrifugation data [8]. Single-molecule fluorescence of PCPs indicated that monomeric and trimeric MFPCP can coexist [9,10]. In this study, we aimed to analyze the oligomeric state of PCP from Symbiodinium. The dinoflagellate Symbiodinium has received increasing attention in recent years because of its significant role

http://dx.doi.org/10.1016/j.febslet.2015.07.039 0014-5793/Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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in coral bleaching [11–16]. Its LHCs, as essential machinery in photosynthesis, are thought to be important in bleaching events [11]. Previous studies showed that Symbiodinium PCP has 80% amino acid sequence similarity to MFPCP of Amphidinium and possesses almost identical spectroscopic properties [17,18]. However, whether it can also form trimers is not clear. Here, we describe our work on the determination of the oligomeric state of Symbiodinium PCP via size-exclusion chromatography (SEC), small angle neutron scattering (SANS) and native mass spectrometry (native MS). SANS is a powerful tool for investigating macromolecules at length scales of 1 nm to 1 lm [19]. Since neutrons are non-destructive, SANS has been widely used to study the structural properties of proteins and protein/membrane interactions, etc. Several studies applied this technique to examine photosynthetic protein complexes of various sources [20–25], including the B820 subunit from Rhodospirillum rubrum LH1 complex, spinach LHC II, Rhodobacter sphaeroides reaction center-cytochrome c2 complexes, as well as chlorosomes, the B808-866 complex, and the reaction center (RC) in the thermophilic green bacterium Chloroflexus aurantiacus. Native MS utilizes nano electrospray ionization (nESI) to generate charged gas phase protein ions directly from aqueous solution, while preserving the integrity of the protein complex. As it is very sensitive, much less time-consuming, and requires a relatively small amount of protein compared to traditional biophysical methods such as X-ray crystallography and NMR, native MS is widely used to analyze protein stoichiometry, topology and dynamics [26–31]. In this work, we analyzed the oligomeric state of PCP in the dinoflagellate Symbiodinium using size-exclusion chromatography (SEC), small angle neutron scattering (SANS) and native mass spectrometry (native MS). SEC and SANS analysis indicated PCP exists as monomers. Native MS demonstrated two oligomeric states of PCP (monomeric and trimeric), with the monomeric PCP being dominant.

Ammonium acetate was chosen to facilitate native MS analysis, as both ammonia and acetic acid are volatile and evaporate readily during electrospray. Elution profiles were recorded using a UV absorbance detector (Bio-Rad) at 280 nm for standards, 280 and 478 nm for PCP. 2.3. Small angle neutron scattering SANS experiments were carried out using the Bio-SANS instrument at the High Flux Isotope Reactor of Oak Ridge National Laboratory [32]. Scattering data were collected at sample-to-detector distances of 0.3 and 6 m to cover a Q range of 0.0067–0.73 Å1 using 6 Å neutron beam (q = (4p/k) sin(h/2), where k is the neutron wavelength and h is the scattering angle). For SANS analysis, PCP samples were prepared by exchanging the buffer from 20 mM Tris–HCl pH 8.0 in H2O to the same buffer in D2O (pD 7.6) using Zeba spin desalting columns (7K MWCO, Thermo Fisher Scientific). Scattering data were recorded at three protein concentrations, 2.7, 5.4, and 8 mg/mL. Data from PCP at 8 mg/mL with additional 150 mM NaCl was also recorded. Appropriate buffer solutions without proteins were collected for background correction. SANS data were reduced and analyzed using IGOR Pro [33]. Data fitting was performed using cylindrical and ellipsoidal models, and the quality of the fitting was evaluated with the reduced v2 parameter [34]. Theoretical scattering curves of PCP monomer and trimer were calculated with the crystal structure of PCP (PDB ID: 1PPR) using CRYSON [35], and fitted to the data. Rg, radius of gyration, was calculated based on Eqs. (1) and (2) for the cylindrical and ellipsoidal models, respectively.

R2g ¼

R2 L 2 þ 2 12

R2g ¼

a2 þ b þ c2 5

ð1Þ

2

ð2Þ

2. Materials and methods

2.4. Native mass spectrometry

2.1. PCP preparation

PCP was concentrated by a MWCO filter (10 kDa, Vivaspin, GE Healthcare) and then buffer-exchanged to 200 mM ammonium acetate with a Micro Bio-Spin 6 chromatography column (MW exclusion limit 6 kDa, Bio-Rad). The final monomer concentration was 5 mg/mL (120 lM). The optimal concentration was adjusted depending on the quality of mass spectra. 5 lL sample was introduced into a quadrupole time-of-flight mass spectrometer (SYNAPT G2 HDMS, Waters) via a gold-coated nanoflow capillary prepared in house [29]. The instrument was operated under gentle electrospray ionization (ESI) conditions (capillary voltage of 1.1–1.5 kV, sampling cone voltage of 60 V, extraction cone voltage of 2 V, and source temperature of 30 °C). The collision energy at the trap and transfer region was adjusted from 8 to 110 V to gradually remove pigments from PCP. The pressure of the vacuum/backing region was 5.1–5.6 mbar. Each spectrum was acquired from m/z 100 to 8000 every 1 s. The instrument was externally calibrated to m/z 8000 with the clusters produced by ESI of a NaI solution. The peak picking and data processing were performed in Masslynx (version 4.1, Waters).

PCP from Symbiodinium sp. CS-156 was prepared according to [17] with minor modifications. Briefly, cells in late exponential phase were harvested by centrifugation at 8000g, resuspended in 50 mM tricine 20 mM KCl (pH 7.5), and broken by three passes through a French pressure cell at 8.3  107 Pa. After removing cell debris and unbroken cells, solid ammonium sulfate was added to 50% saturation to precipitate unwanted proteins. The resulting supernatant was dialyzed against 20 mM Tris–HCl (pH 8.0), concentrated, filtered through a 0.2 lm filter, and applied to a HiTrap™ Q Sepharose™ HP column (GE Healthcare) and eluted with a linear gradient of NaCl from 0 to 0.5 M in the same buffer. Fractions with highest A478nm:A280nm ratios (greater than 4.1) were pooled, and applied to a HiLoad™ Superdex™ 200 prep grade column (GE Healthcare). 2.2. Size-exclusion chromatography SEC was performed on a Bio-rad FPLC system with a Superdex 200 10/300 GL column (GE Healthcare), which was calibrated with bovine serum albumin (monomeric BSA, 67 kDa; dimeric BSA, 134 kDa), b-lactoglobulin (BLG, 36.6 kDa), and ribonuclease A (RNase A, 13.7 kDa). Blue dextran (2 MDa) was used to determine the void volume of the column. All standards were purchased from Sigma Aldrich. 200 mM ammonium acetate (without adjusting the pH) was used as the mobile phase at a flow rate of 0.4 mL/min.

3. Results and discussion 3.1. Size-exclusion chromatography A Superdex 200 10/300 GL column (GE Healthcare) was calibrated with BSA, BLG and RNase A (Fig. 1). The MW of PCP was determined to be 41 kDa, which is consistent with the size

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A 280nm

Normalized A 280nm

J. Jiang et al. / FEBS Letters 589 (2015) 2713–2719

1.2 1.0 0.8 0.6 0.4 0.2 0.0 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

BSA

Blue Dextran

RNase A BLG BSA dimer PCP

5

10

15

20

Elution volume (mL) Fig. 1. SEC analysis of PCP in 200 mM ammonium acetate. Top: elution profile of the void volume marker Blue Dextran (elution volume Ve 7.7 mL) and standards (Ve 12.1, 13.8, 15.1 and 17.3 mL for BSA dimer, BSA, BLG and RNase A, respectively). Bottom: elution profile of PCP (Ve 14.8 mL. The running conditions were the same. Based on the linear relationship between Ve and Log10 (MW), which was Log10 (MW) = 0.1914 Ve + 4.453 (R2 = 0.9994), the average molecular mass of native PCP was determined to be 41 kDa.

calculated based on the crystal structure of Amphidinium monomeric PCP (one polypeptide (32.7 kDa [17]), eight peridinins (630.81 Da), two Chl a (893.49 Da) molecules and two DGDG (949.3 Da) molecules in each 1PPR monomer, sum of 41.4 kDa). Analysis of the mass of PCP in non-volatile buffers (e.g. 20 mM sodium phosphate pH = 7 with or without 150 mM NaCl, 20 mM bis-Tris pH = 6, 6.5 with or without 150 mM NaCl) demonstrated that different buffers did not change the oligmerization state of protein (data not shown). 3.2. Small angle neutron scattering To maximize the protein scattering and lower the background scattering, PCP proteins were exchanged from H2O buffer into D2O buffer prior to the SANS experiments, because deuterium atoms have much lower incoherent scattering, which contributes to the background signal. In order to test whether D2O would alter the structure of PCP, circular dichroism (CD) spectra of PCP in the UV/Vis region were taken at 30 min, 2 h and 1 day after the buffer exchange. No significant differences were observed between the CD spectra of PCP in H2O and D2O buffers (data not shown), suggesting that the structure of PCP stays the same in D2O as in H2O. SANS experiments were carried out with PCP at three concentrations, 2.7, 5.4 and 8 mg/mL (Fig. 2). The three profiles showed essentially the same features, indicating there was no concentration dependent effect on the shape of the scattering curves. The increase in scattering intensity in the low Q region is consistent with the presence of large scattering objects such as large protein aggregates that could not be dispersed by the addition of 150 mM NaCl. Due to the absence of a plateau in low Q region, it was not possible apply Guinier analysis. However, it was possible to obtain structural information about the system by fitting ellipsoidal and cylindrical geometric shapes to the SANS data in the Q range 0.03 < Q < 0.36 Å1 (cylinder: Table 1 and Fig. 3, ellipsoid: Fig. S1). The fits to all four SANS profiles are shown in Fig. 2. Results of fitting were consistent: the calculated values of Rg (radius of gyration) based on both models (cylinder: 19.9–20.9 Å, ellipsoid: 18.5–19.2 Å) were similar to the calculated Rg of monomeric PCP (20.0 Å), suggesting that PCP existed as monomers in solution. As an example, the fitting of data from 2.7 mg/mL PCP using a cylindrical model was shown in Fig. 3.

Fig. 2. SANS profiles of PCP in D2O buffer. Curves are offset for clarity.

Table 1 Structural parameters obtained from SANS data by cylindrical model. Rg for the crystal structure (PDB ID: 1PPR) was calculated in CRYSON [35]. Cylinder radius R (Å) Crystal structure, monomer Crystal structure, trimer 8 mg/mL 5.4 mg/mL 2.7 mg/mL 8 mg/mL with 150 mM NaCl

Cylinder length L (Å)

Radius of gyration, Rg (Å)

v2

20.0 33.5 16.9 ± 0.02 16.9 ± 0.03 16.6 ± 0.04 16.9 ± 0.03

55.1 ± 0.2 55.1 ± 0.2 59.8 ± 0.3 56.7 ± 0.3

19.9 19.9 20.9 20.3

15.1 11.6 8.3 10.3

In addition, comparison of the experimental scattering curve of Symbiodinium PCP with scattering profiles calculated from monomeric and trimeric Amphidinium PCP based on the crystal structure (Fig. 3A) also shows that the monomeric conformation is favored over the trimer. We then wanted to test whether a mixture of monomeric and trimeric PCP could better represent the data. Based on the result from the program Oligomer [36], 100% monomeric PCP fitted best to the data (0.03 < Q < 0.36 Å1). However, this analysis seemed to be insensitive to up to 10% of trimeric PCP while using the theoretical scattering profiles of monomeric and trimeric PCP as standards. In summary, all the analysis suggests that PCP is largely monomeric under the tested conditions. 3.3. Native mass spectrometry To determine the oligomeric state(s), the mass of PCP was measured by native MS. PCP molecules, solvated by ammonium acetate and water, were introduced into the gas phase by gentle nanoESI. PCP of the initial concentration (120 lM) was too viscous to spray, we therefore gradually added 200 mM ammonium acetate to dilute the sample. The final protein concentration was about 50 lM ( 2 mg/mL). In order to remove the associated unwanted molecules and release the intact protein assembly, we applied collision activation. By ramping the collisional energy (8 V), we observed two series of charged states, representing two species with a MW of 41.3 kDa (charge states 12+ to 14+) and 123 kDa (charge states 22+ to 24+), respectively (Fig. 4). The former was assigned to PCP monomers, and the latter to the trimeric PCP. Native MS demonstrated two oligomeric states of PCP, although the population of PCP trimers was not as significant as that of monomers. It is important to note that we also observed the loss of one or two peridinins from monomeric PCP. This could represent

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Fig. 3. Fitting of SANS data. (A) SANS data from 2.7 mg/mL PCP (green) are fitted by PCP monomer (magenta), PCP trimer (blue), and a cylindrical model (red). Profiles of PCP monomer and trimer were calculated based on the crystal structure (PDB ID: 1PPR) using CRYSON [35]. (B) PCP monomer fits to a cylindrical model. Cylinder radius: 16.6 Å; length: 59.8 Å.

+13

+12

+13

100

+12 +12

80

+13 60

+11 +11

Monomeric PCP Trimeric PCP Monomeric PCP lost one peridinin Monomeric PCP lost two peridinin

%

+14 +14

40

+24

+23

+22

20

0 2000

3000

4000

5000

6000

m/z

Fig. 4. Native MS spectrum of PCP. Protein concentration is 2 mg/mL. Collision energy is 8 V.

the natural heterogeneity of the PCP protein, similar to HSPCP in Amphidinium [6]. When the collision energy of the mass spectrometer trap region was increased, substantial loss of bound pigments from the protein complex was detected, allowing us to estimate the relative strengths of non-covalent interactions between cofactors and the PCP polypeptide. At 68 V, three charged series (Fig. 5A) were observed, corresponding to the partially denatured PCP containing two Chl a molecule and one peridinin, two Chl a molecules, one Chl

a molecule, respectively, with the first species having the most intense signal. At this point, no trimers or trimers with partial cofactors were identified, indicating the interactions between monomers were not strong. When the collision energy continued to rise to 108 V, more pigments dissociated from their binding sites, resulting in the polypeptide with no pigments (Fig. 5B). These results indicate that Chl a molecules in PCP has stronger non-covalent interactions with the polypeptide chain compared to peridinins and lipids, and thus are more resistant to unfolding.

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+12 Protein+2xChl a +1xPeridinin

A

Protein+2xChl a 2883.3

100

Protein+1xChl a

+12 +12 +11

+13 2947.8

+13 %

+13

3149.3

+11

3065.3

+11 3218.5

2662.8 2809.7

3286.6 3374.3

2721.9 2591.1

2460.2 2407.4

0

2400

2520.0

2500

2600

2700

2800

2900

3000

3100

3200

3300

3400

3500

3600

3700

3800

3900

4000

m/z 4100

B 893.6

100

Protein

+12 %

2734.0

+11 2982.1

615.3

+13 2523.8

+10 3281.9

1787.1 800.6

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

2600

2800

3000

3200

3400

3600

3800

m/z 4000

Fig. 5. Dissociation of PCP with a collision energy ramp. Protein concentration is 2 mg/mL. Collision energy: (A) 68 V, (B) 108 V.

The observation is in agreement with the pigment arrangement in the PCP crystal structure (1PPR): two Chl a molecules sit in the central of the complex, surrounded/protected by peridinins and lipids. In addition, when PCP was fully unfolded (Fig. 5B), the protein carried 10–13 positive charges, which were significantly fewer compared to PCP apoprotein under regular LC/MS conditions (21+ to 41+ charges [17]). This is because in native MS, protein molecules are solvated in ammonium acetate at near-neutral pH instead of the water/acetonitrile/formic acid mixture at pH 1–2 in regular LC/MS, where protons are more abundant. Chl a and peridinin were also observed, with the Chl a molecular ion being the base peak. This does not mean that Chl a is more abundant than

peridinin in the complex, as ionization efficiencies of the two molecules could be very different, which was the case in our experiment. We were unable to identify DGDG, which probably was poorly ionized or underwent fragmentation. In this study, we showed the co-existence of monomeric and trimeric PCP. Collision-induced dissociation of PCP indicated that the Chl a molecules have stronger non-covalent interactions with the polypeptide chain compared to peridinins and lipids, and thus are more resistant to unfolding. Our results demonstrate native MS as a powerful complementary tool to X-ray crystallography and NMR in biophysical studies. Although it cannot offer high resolution data, it interrogates protein complexes in their near-native

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state with small amounts of samples and fast turnaround [31], and has provided unique insights into the photosynthetic apparatus, which is highly dynamic and often lacks crystal structures [26,28,37]. 3.4. The functional oligomeric state of PCP Previous studies on PCP mostly focused on MFPCP of Amphidinium, the structure of which has been resolved to 2.0 Å by X-ray crystallography. There is some indirect evidence linking the trimerization to the function of this protein complex. The trimeric MFPCP was thought to be relevant for the in vivo oligomeric state in the thylakoid lumen based on unpublished analytical ultracentrifugation data [8]. Single molecule fluorescence of PCPs indicated that monomeric and trimeric MFPCP could coexist in certain but not all buffers while recombinant PCP was always monomeric [9]. The histogram of fluorescence emission intensities for MFPCP in Tris buffer showed two clear peaks. The peak of the lower intensity was assigned to monomers, while another peak, which appeared at about 2.5 times higher intensity, was due to the trimeric form [9]. In another single-molecule study under dilute conditions, more than 99.9% of PCP was monomeric, but very rarely, objects with triple the monomeric intensity were observed, implying the existence of trimers [10]. A piece of indirect evidence suggesting PCPs of these two genera could adopt different oligomeric states was the distinct lifetimes of long-wavelength absorbing Chl a [18]. MFPCP from Amphidinium had a lifetime ranging between 2 and 4.5 ns [38–40], which is substantially shorter than 77.4 ns measured in the Symbiodinium PCP [18]. The shortening of the lifetime in MFPCP may be associated with a slow phase of annihilation of Chl a excited states occurring between PCP trimers even at low excitation intensities [38]. However, discrepancies could simply originate from non-unified methodologies (different temperatures, fitting models, exciting laser intensities, etc.) to perform experiments and analyze spectroscopic data, instead of different oligomeric states of two PCPs [18]. Calculations of Förster energy transfer rates in MFPCP indicated the two anisotropy decay time constants of 6.8 ± 0.8 ps and 350 ± 15 ps corresponded to intraand intermonomeric excitation equilibration times, respectively. The significantly longer life time meant that the intermonomeric energy transfer in vivo, if it existed, was substantially slower that the faster energy transfer within monomers [41]. In our study, native MS demonstrated two oligomeric states of PCP, with the dominance of monomeric PCP, but we know that Symbiodinium PCP also crystalizes as trimers (unpublished, E. Hoffman, personal communication). Therefore, amino acid sequence variations may not be the sole source of different oligomeric states of two PCPs (Fig. S2, Symbiodinium PCP has 80% amino acid sequence similarity to MFPCP of Amphidinium). From the characterization done by our group, Symbiodinium PCP possesses very similar spectroscopic properties (steady-state absorption and fluorescence [17], femto- and nanosecond time-resolved absorption [18], picosecond time-resolved fluorescence [18], CD compared to its Amphidinium counterpart. Its function as a light-harvester does not alter though it is still mainly monomeric even at very high concentrations (up to 8 mg/mL). Therefore, we conclude that the trimerization is not necessary for PCP to function as a light-harvesting complex. Acknowledgements The authors would like to thank Drs. David Kramer and Atsuko Kanazawa of Michigan State University for providing the Symbiodinium culture, and Ms. Mindy Prado for helping with cell culturing. This research is from the Photosynthetic Antenna Research Center (PARC), an Energy Frontier Research Center

funded by the DOE, Office of Science, Office of Basic Energy Sciences under Award Number DE-SC 0001035. The Center for Structural Molecular Biology operates Bio-SANS and is supported by the U.S. DOE, Office of Science, Office of Biological and Environmental Research under FWP ERKP291. The High Flux Isotope Reactor is sponsored by the Scientific User Facilities Division, Basic Energy Sciences. Oak Ridge National Laboratory (ORNL) is managed by UT-Battelle, LLC, for the U. S. Department of Energy (DOE) under contract No. DE-AC05-00OR22725. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2015.07. 039. References [1] Haxo, F.T., Kycia, J.H., Somers, G.F., Bennett, A. and Siegelman, H.W. (1976) Peridinin-chlorophyll a proteins of the dinoflagellate Amphidinium carterae (Plymouth 450). Plant Physiol. 57, 297–303. [2] Prezelin, B.B. and Haxo, F.T. (1976) Purification and characterization of peridinin-chlorophyll a proteins from marine dinoflagellates Glenodinium sp. and Gonyaulax polyedra. Planta 128, 133–141. [3] Hofmann, E., Wrench, P.M., Sharples, F.P., Hiller, R.G., Welte, W. and Diederichs, K. (1996) Structural basis of light harvesting by carotenoids: peridinin-chlorophyll-protein from Amphidinium carterae. Science 272, 1788– 1791. [4] Hiller, R.G., Broughton, M.J., Wrench, P.M., Sharples, F.P., Miller, D.J. and Catmull, J. (1999) Dinoflagellate light-harvesting proteins: genes, structure and reconstitutionThe Chloroplast: From Molecular Biology to Biotechnology, pp. 3–10, Springer, Netherlands. [5] Norris, B. and Miller, D. (1994) Nucleotide sequence of a cDNA clone encoding the precursor of the peridinin-chlorophyll a-binding protein from the dinoflagellate Symbiodinium sp. Plant Mol. Biol. 24, 673–677. [6] Schulte, T., Sharples, F.P., Hiller, R.G. and Hofmann, E. (2009) X-ray structure of the high-salt form of the peridinin-chlorophyll a-protein from the dinoflagellate Amphidinium carterae: modulation of the spectral properties of pigments by the protein environment. Biochemistry 48, 4466– 4475. [7] Schulte, T., Niedzwiedzki, D.M., Birge, R.R., Hiller, R.G., Polivka, T., Hofmann, E. and Frank, H.A. (2009) Identification of a single peridinin sensing Chl-a excitation in reconstituted PCP by crystallography and spectroscopy. Proc. Natl. Acad. Sci. 106, 20764–20769. [8] Polívka, T. and Hofmann, E. (2014) Structure–function relationship in peridinin-chlorophyll proteinsThe Structural Basis of Biological Energy Generation, pp. 39–58, Springer, Netherlands. [9] Wormke, S., Mackowski, S., Schaller, A., Brotosudarmo, T.H., Johanning, S., Scheer, H. and Brauchle, C. (2008) Single molecule fluorescence of native and refolded peridinin-chlorophyll-protein complexes. J. Fluoresc. 18, 611–617. [10] Bockenhauer, S.D. and Moerner, W.E. (2013) Photo-induced conformational flexibility in single solution-phase peridinin-chlorophyll-proteins. J. Phys. Chem. A 117, 8399–8406. [11] Takahashi, S., Whitney, S., Itoh, S., Maruyama, T. and Badger, M. (2008) Heat stress causes inhibition of the de novo synthesis of antenna proteins and photobleaching in cultured Symbiodinium. Proc. Natl. Acad. Sci. 105, 4203– 4208. [12] Takahashi, S., Whitney, S.M. and Badger, M.R. (2009) Different thermal sensitivity of the repair of photodamaged photosynthetic machinery in cultured Symbiodinium species. Proc. Natl. Acad. Sci. 106, 3237–3242. [13] Rowan, R. (2004) Coral bleaching: thermal adaptation in reef coral symbionts. Nature 430. 742-742. [14] Warner, M.E., Fitt, W.K. and Schmidt, G.W. (1999) Damage to photosystem II in symbiotic dinoflagellates: a determinant of coral bleaching. Proc. Natl. Acad. Sci. 96, 8007–8012. [15] Iglesias-Prieto, R., Matta, J.L., Robins, W.A. and Trench, R.K. (1992) Photosynthetic response to elevated temperature in the symbiotic dinoflagellate Symbiodinium microadriaticum in culture. Proc. Natl. Acad. Sci. 89, 10302–10305. [16] Roth, M.S. (2014) The engine of the reef: photobiology of the coral-algal symbiosis. Front. Microbiol. 5. [17] Jiang, J., Zhang, H., Kang, Y., Bina, D., Lo, C.S. and Blankenship, R.E. (2012) Characterization of the peridinin-chlorophyll a-protein complex in the dinoflagellate Symbiodinium. Biochim. Biophys. Acta 1817, 983–989. [18] Niedzwiedzki, D.M., Jiang, J., Lo, C.S. and Blankenship, R.E. (2013) Lowtemperature spectroscopic properties of the peridinin-chlorophyll a-protein (PCP) complex from the coral symbiotic dinoflagellate Symbiodinium. J. Phys. Chem. B 117, 11091–11099. [19] Urban, V.S. (2002) Small-Angle Neutron Scattering. In Characterization of Materials, John Wiley & Sons Inc..

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