Elucidating the Molecular Mechanism of Dynamic Photodamage of Photosystem II Membrane Protein Complex by Integrated Proteomics Strategy

Open AccessCCS ChemistryRESEARCH ARTICLE23 Feb 2021Elucidating the Molecular Mechanism of Dynamic Photodamage Photosystem II Membrane Protein Complex by Integrated Proteomics Strategy Ye Zhou, Zheyi Liu, Mingdong Yao, Jun Chen, Yanan Xiao, Guangye Han, Jian-Ren Shen and Fangjun Wang Zhou CAS Key Laboratory Separation Sciences for Analytical Chemistry, Dalian Institute Chemical Physics, Chinese Academy Sciences, 116023 Google Scholar More articles this author , Liu Yao State Catalysis, Systems Bioengineering, Ministry Education, Tianjin University, 300072 Chen Science Technology on Surface Physics Chemistry Laboratory, Jiangyou 621908 Xiao University Beijing 100049 Photosynthesis Research Center, Photobiology, Botany, 100093 Han Interdisciplinary Science, Graduate School Natural Technology, Okayama 700-8530 *Corresponding author: E-mail Address: [email protected] https://doi.org/10.31635/ccschem.021.202000583 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesTrack Citations ShareFacebookTwitterLinked InEmail (PSII), as a multiple-subunit chloroplast membrane-associated pigment-protein complex thylakoid membrane, is primary target light-induced photodamage. However, overall molecular details conformation composition dynamics PSII photodamage are still controversial. In study, we investigated systematically dynamic conformation, degradation, oxidation processes integrating chemical cross-linking top-down proteomics strategies. The disassembly complex, well degradation fragments from both extrinsic intrinsic protein subunits, were feasibly probed during loss O2-evolving activity. Some long-term controversial issues, including activity occurs before detachment Mn4CaO5 cluster, clarified. Finally, detailed route map was outlined at level first time, which markedly enhanced our understanding mechanism Download figure PowerPoint Introduction photosynthetic process in cyanobacteria, algae, plants converts solar energy into generates oxygen water, indispensable sustaining aerobic lives earth. membrane pigment–protein light-dependent reactions photosynthesis photosystem reduces bound plastoquinones catalyzes light-driven water O2.1,2 core composed over 20 different embedded membranes mainly dimer.3 Early biochemical investigations demonstrated that least seven major viz, D1, D2, CP47, CP43, PsbE, PsbF, PsbI, essential evolution activity.4 Although light source photosynthesis, excess will induce apparatuses. damage targeted leads inactivation electron transport with subsequent oxidative oxygen-evolving reaction center.5,6 Many spectroscopy methodologies, fluorescence UV–vis spectra,7,8 paramagnetic resonance (EPS),9 circular dichroism (CD),10 mass spectrometry (MS),11 have been applied monitor photoinactivation elucidate several hypotheses proposed, initiation progress unclear,12 because great challenge monitoring structural typographical changes large conventional methods. recent years, MS-based strategy has promising tool probing composition, assembly, highly complementary biology tools, such X-ray diffraction (XRD) cryo-electron microscopy (cryo-EM).13–15 MS (CX-MS) popular used mapping protein–protein interactions dissecting assembly structure complexes.16–18 Recently, CX-MS analyses revealed PsbQ subunit location cyanobacterial complex,19 provided evidence multiple between PsbP subunits spinach complex.20,21 On other hand, analysis intact proteins rapidly developed powerful complexes,22,23 plant proteomics,24,25 exhibits distinct advantages characterizing proteoforms.26,27 Herein, (CX) strategies ( Supporting Information Figure S1). results obtained these two high complementarity could validate each other. along process. Briefly, specific residues around accompanied susceptible D1 D2. accumulation induced finally, entire part lumen-side domain disassembled. Our demonstrate crucial roles loss, providing pieces process, thereby enhancing fundamental Experimental Methods Preparation Cyanobacterial purified thermophilic cyanobacterium Thermosynechococcus vulcanus, described previously.28,29 Further, 1 mM 6-aminocaproic acid benzamidine added sample preparation buffers avoid unexpected endogenous enzymes. suspended buffer containing 30 Mes (pH 6.0), NaCl, 3 CaCl2 exposed under saturating white illumination 0, 15, 60, 180, 300 min 4 °C, respectively. Steady-state measured using Clark-type electrode 25 °C red illumination.28,29 corresponding 5 μg chlorophyll/mL dispersed 0.60 recrystallized 2,6-dichlorobenzoquinone, 10 CaCl2, rate via O2 concentration. Cross-linking samples 50 N-2-hydroxyethylpiperazine-N-2′-ethanesulfonic (HEPES; pH 7.4) diluted μg/μL. For cross-linking, incubated cross-linker bis(sulfosuccinimidyl)suberate (BS3; 1:1, w/w) room temperature h NH4HCO3 terminate reaction. All precipitated five volumes ice-cold precipitation solution (ethanol/acetone/acetic = 50:50:0.1) −20 overnight. resuspended denaturing 8.0) 6 M guanidine Tris. After reduction tris(2-chloroethyl)phosphate (TCEP; thiol-free reducing agent) alkylation iodoacetamide (IAA) 15 dark, 2 Tris digested chymotrypsin 16 37 an enzyme substrate ratio 1/25 (w/w). peptide mixtures C18 solid-phase extraction (SPE) column liquid chromatography (LC)-MS/MS analysis. Online SCX-RPLC-MS/MS online strong cation exchange (SCX)-reversed-phase LC (RPLC)-MS/MS analysis, cm SCX (200 μm i.d.) prepared previously described30 trapping first-dimension separation column. A series stepwise elution salt concentrations 150, 250, 350, 450, 1000 NH4AC elute peptides second-dimensional capillary (75 μm, i.d., length) packed particles (3 120 Å). Each lasted followed equilibrium 0.1% formic (FA) additional min. step RPLC-MS/MS 100 gradient 5% 35% acetonitrile (ACN). flow nL/min. Solutions FA ACN mobile phases B, An LTQ-Orbitrap Velos spectrometer (Thermo, San Jose, CA) perform proteolytic samples. parameters set follows: ion transfer 250 spray voltage 2.0 kV, full scan m/z 350 1800 resolution 30,000 centroid mode. Data-dependent MS/MS scans R 7500 performed selecting most intense ions higher-energy collisional dissociation (HCD) normalized collision energy. carrying +1, +2, or unknown-charges excluded MS2 scans. exclusion repeat count 1, duration s, s. Top-down LC-MS/MS levels initially redissolved 70% FA, precooled °C,31 incubation six injected immediately instrument loaded C5 trap separated 80% ACN/0.1% utilized B. eluted 20–100% phase B 150 min, isocratic 100% Q-Exactive hybrid quadrupole-Orbitrap (Thermo) analyses. 320 500 2500 120,000. HCD 25%. also acquired Orbitrap analyzer 60,000, automatic gain control (AGC) × 106 max injection time ms. charge <+2 excluded, 180 data sequences within UniProt http://www.uniprot.org/). Cross-linked identified pLink software,32 its search instrument, HCD; precursor tolerance, ppm; fragment BS3 (cross-linking sites K N-terminus, cross-link mass-shift 138.068 Da, monolink 156.079 Da); fixed modification carbamidomethyl cysteine (+57.02146 variable methionine (+15.99492 length, minimum amino acids maximum 60 per chain; mass, 600 6000 Da enzyme, chymotrypsin, up missed cleavage cross-link. filtered, requiring false discovery (FDR) < scoring matches (SVM) score ≥1. spectra annotated pLabel, deviation ≤20 ppm. Mapping cross-links models Pymol, schematic visualizations generated xiNET.33 RAW files searched against ProSightPC (version 4.0.2.1; Thermo) Thrash algorithm. Intact masses logic tree submitted all absolute search, biomarker did not yield ID expected hit (E) value less than 1E-4 search. precursors >750 more searched. tolerances ppm, FDR 1% controlled E below To identify oxidized residues, MSPathFinder 1.0.6510).34 Mass error tolerance ppm fragments. types modifications allowed sequence. included methionine/tryptophan/phenylalanine/leucine/aspartate/glutamate oxidation, dehydrogen, N-terminal formylation. Other minimum–maximum (min–max) sequence lengths 21–300, min–max charges 2–30, 1–15, 3000–50,000. ≤1E-4 Q-value ≤0.01. deposited ProteomeXchange Consortium PRIDE35 partner repository dataset identifier PXD021369. Results Discussion [over 2000 μmol (mg chlorophyl)−1 h−1] purified, Five aliquots respectively, S2). varying chemically cross-linked BS3, covalently link proximal lysine 30-Å distance.36,37 Then total 1928 be matched 218 pairs (FDR 5%, SVM ≥ 1; Tables S1 It noteworthy 45 stably existed whole (Figure 1a Table S1), among 35 mapped crystal (Cα–Cα distance ≤ Å; Figures 1b 1c). stable whose local regions might relatively course Specifically, structure-stable came those K, O, U, V S3a). PsbE located C-terminal region, close proximity cluster. Interestingly, almost PsbO, belonged ones. supporting maximal physiological conditions.38,39 Therefore, main even though lost. | (a) shown schematically boxes. stromal domain, transmembrane, lumenal presented yellow, pink, blue, Teal lines purple arcs indicate inter intra cross-links, (b) monomeric Å, PDB code: 3WU2). distances four due unresolved structure, appear incompatible model (30 Å Cα–Cα 41 These attributed nonspecific native alternative conformations structure. denoted deep blue Å) firebrick lines. colored pale green, salmon, olive, bright orange, teal, lysines spheres colors. cluster sphere. (c) spectrum CP43/GANVGSAQGPTGLGKY-PsbO/ARANVKRF. CP43/317K-PsbO/186K. Peaks α- β-peptides contrast, 27 only remaining unstable Hence, domains altered significantly Most structure-unstable come small PsbH, I, M, X, Z. CP47 belong stroma-side while region S3b). suggested significant CP43 stroma side lumen side. Since plays critical role coordinating any may result reaction, explain caused Furthermore, D2 one center conformation/structure cause oxidation. products CP47/136K-D1/310K, PsbM/34K-D1/238K, CP47/372K-D2/254K disappeared very early stage (0–15 min; S4 S2), indicated occurred Part related illumination. example, CP47/388K-PsbU/24A PsbO/186K-PsbU/1A vanished when exceeded S4). further PsbO disassembled Meanwhile, some new emerged after 180-min illumination, CP43/301K-PsbH/19K, PsbO/192K-PsbY/1E, CP43/193K-PsbX/35K S4), suggest photodamage-induced structure/conformation re-arrangement regions. Overall, above outline rough changes/degradation especially subunit. by, simultaneously degradations/conformation alternations CP43. structural/conformational observed, leading complete electron-transfer reactions. detail, subunits. We characterized S5) compared intensity 11 S3). their weights (>40 kDa) difficult analysis.40,41 Along F, H, L, T, Z decreased accordingly, indicating PsbU alter consistent discoveries stable, whereas PsbH I partially degraded Besides, there upward trends (1G-14D), (439R-451D), (324Q-342L), (69R-84K), (140F-156D, 225A-244A), (1A-14D), PsbV (144H-163Y) (Figures 2a–2c). Among these, 2a) contribute directly evolution. (1G-14D) (439R-451D) 2a). involved vicinity S6). D1/310K PsbV/150K PsbE/69R-84K. CP43/435K C terminus 439R-451D. D2/13K PsbX/35K PsbF. D2/307K PsbO/51K 140F-156D 225A-244A possibly supported appearances increased presented. L lime, slate, smudge, aquamarine, (140F-244A), darker shades CP47/1G-14D. b y green reactive species (ROS)-induced believed photoinhibition turnover living cells.42,43 studies discovered subunits,44 nearby QA PheoD1 subunits,45,46 S7). whether ROS controversial.47 3a 3b S5).43 During state without found oxidized, CP43/447M, D2/97L, D2/329F. 15-min 13% extra D2/304F D2/311L, oxidized. C-terminus photoinduced occur metal 60-min 29% D2/309L, 310L, 318W, 321P numerous D2/256W, 269L, 299P, 319M, 323D, D2/299P, 309L, 321P, 323D suggesting they deactivation aforementioned D2/256W 269L particularly interesting. Also, PheoD2 256W nonheme iron, implying occurrence acceptor 300-min observed D2/161P, 171F, 172L, 173L, 325P; CP47/15P, 18L, 49P, 51L; CP43/103L, 124F, 445L, 447M. near distributed complex. Additionally, later stages photo