Washington University in St. Louis Washington University Open Scholarship Arts & Sciences Electronic Theses and Dissertations Arts & Sciences Winter 12-15-2016 Investigating the Life-cycle of Photosystem II Using Mass Spectrometry Daniel Avram Weisz Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/art_sci_etds Recommended Citation Weisz, Daniel Avram, "Investigating the Life-cycle of Photosystem II Using Mass Spectrometry" (2016). Arts & Sciences Electronic Theses and Dissertations. 1009. https://openscholarship.wustl.edu/art_sci_etds/1009 This Dissertation is brought to you for free and open access by the Arts & Sciences at Washington University Open Scholarship. It has been accepted for inclusion in Arts & Sciences Electronic Theses and Dissertations by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY Department of Chemistry Dissertation Examination Committee: Michael L. Gross, Co-chair Himadri B. Pakrasi, Co-chair Robert E. Blankenship Dewey Holten Joseph Jez Investigating the Life-cycle of Photosystem II Using Mass Spectrometry by Daniel Avram Weisz A dissertation presented to The Graduate School of Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy December, 2016 Saint Louis, Missouri © 2016, Daniel Weisz Table of Contents List of Figures………………………………………………………………………......................v List of Tables……………………………………………………………………………………viii Abbreviations……………………………………………………………………………………..ix Acknowledgements……………………………………………………………………………….xi Dedication……………………………………………………………………………………….xiv Abstract…………………………………………………………………………………………..xv Chapter 1: Introduction: The use of advanced mass spectrometry to dissect the life-cycle of Photosystem II……..………………………………………..……..….1 Summary………………………….……………………………………………….2 Introduction……………………………………….……………………………….2 Composition of PSII complexes…………………………………………………..7 MS-based methods to study the composition of PSII complexes………....7 PSII life-cycle application: Composition of subcomplexes……………...13 PSII life-cycle application: Identification of accessory proteins………...20 PSII life-cycle application: Identification of PTMs……………………...22 Dynamics: Quantitative or semi-quantitative changes in PSII proteins and PTMs……………………………………………………………………………..27 MS-based methods to study PSII dynamics……………………………...27 PSII life-cycle application: Measuring changes in phosphorylation levels……………………………………………………………………..32 PSII life-cycle application: Measuring changes in oxidation levels……..34 PSII life-cycle application: Measuring the temporal dynamics of life- cycle events using isotopic labeling……………………………………...35 Structure: Determining protein-protein interactions in PSII complexes…………37 MS-based methods to study PSII structure………………………………37 PSII life-cycle application: Cross-linking and footprinting to determine interactions among PSII subunits….……………………………………..40 This work………………………………………………………………………...47 References…..……………………………………………………………………51 ii Chapter 2: Structural analysis of Photosystem II: Mass spectrometry-based cross-linking study shows that the Psb28 protein binds to cytochrome b559…..72 Summary………………………………………………………………………....73 Introduction………………………………………………………………………74 Results……………………………………………………………………………76 Discussion………………………………………………………………………..84 Materials and Methods……………………………………………………..…….93 References…………………………………………………………………........100 Supplementary Results and Discussion…………………………………….…..106 Chapter 3: Oxidative modifications of PSII detected by mass spectrometry……….…….121 Summary………………………………………………………………………..122 Introduction……………………………………………………………………..122 Results and Discussion…………………………………………………………124 Materials and Methods……………………………………………………….…144 References………………………………………………………………………147 Chapter 4: Structural localization of PsbQ in Photosystem II using chemical cross-linking and mass spectrometry…...………………………………………149 Summary………………………………………………………………………..150 Introduction……………………………………………………………………..150 Results…………………………………………………………………………..152 Discussion………………………………………………………………………169 Materials and Methods………………………………………………………….178 References………………………………………………………………………183 Chapter 5: Conclusions, future directions, and additional work………………………......194 Summary and conclusions of this work………………………………………...195 Future directions for MS contributions to PSII life-cycle research…………….197 General conclusion…………………………….………………………………..199 Additional work: Preliminary characterization of a PSII complex lacking the RC subunits…………………………………………………………………201 iii Results and Discussion…………………………………………………201 Materials and Methods………………………………………………….204 References………………………………………………………………205 iv List of figures Chapter 1: Introduction: The use of advanced mass spectrometry to dissect the life-cycle of Photosystem II Figure 1 Publications that use MS for PSII research, 1979-2015……………...…………..4 Figure 2 A schematic of the PSII life-cycle……………………………...………………..14 Chapter 2: Structural analysis of Photosystem II: Mass spectrometry-based cross-linking study shows that the Psb28 protein binds to cytochrome b559 Figure 1 Screening of mutant strains for elevated Psb28 content, characterization of the ΔpsbO PSII monomer, and quantification of Psb28 content in ΔpsbO PSII………………………………………………………………………………77 Figure 2 Immunoblot comparing ΔpsbO-PSII before and after cross-linking with 50, 100, and 300 molar excess BS3:PSII…………..……………………..………79 Figure 3 Mass spectrometric data showing a cross-link between (A) Psb28-K8 and PsbE-S2 and (B) Psb28-K8 and PsbF-A2…………...…………..............……….82 Figure 4 Binding model of Psb28 to RC47 based on cross-linking results……...………...86 Figure 5 A schematic of the PSII assembly process………………..……………………..92 Figure S1 Distribution of linear (Euclidean) distances between C-α’s of cross-linked residues in ΔpsbO-PSII........................................................................................113 Figure S2 Mass spectrometric data showing a cross-link between Psb28-A2 and PsbF- A2…………………………………………………………………………….....114 Figure S3 Intact-mass spectra of PsbE, PsbF, and Psb28……….…………………………115 Figure S4 PsbE-Psb28 cross-link detected in His47 sample………………………………116 Figure S5 PsbF-Psb28 cross-link detected in His47 sample……………………………….117 Figure S6 Isotope-encoded PsbF-Psb28 cross-link detected in His47 sample….…………118 Figure S7 Top docked conformations of Psb28 to RC47……………….…………………119 Figure S8 Psb28 binding above the PSII cytosolic-surface cavity……………….………..120 Chapter 3: Oxidative modifications of PSII detected by mass spectrometry Figure 1 Examples of MS/MS spectra detected that identify oxidative modifications of PSII residues……………...…......……...……………………………………126 v Figure 2 Residues with oxidative modifications detected in this study………...………..134 Figure 3 Cytosolic-side oxidative modifications detected in this study………………….135 Figure 4 Lumen-side oxidized residues detected in this study………………...…………137 Figure 5 Comparison of the lumen-side oxidative modified residues with channels previously identified in PSII by computational studies……...…….….139 Figure 6 Comparison of lumen-side oxidized residues detected by Frankel et al. (2012) and the current study……………………………………...141 Chapter 4: Structural localization of PsbQ in Photosystem II using chemical cross-linking and mass spectrometry Figure 1 Phylogenetic tree showing evolutionary relationship between PsbQ protein sequences from selected cyanobacterial species……...……..…………..153 Figure 2 Native-gel and SDS-PAGE analysis of HT3-PSII and Q-His-PSII…………...………...154 Figure 3 Chemical cross-linking of PsbQ with other PSII subunits…………...………………….156 Figure 4 Product-ion (MS/MS) spectra obtained for cross-linked peptides……...……...………..158 Figure 5 Gel electrophoresis and immunoblot analysis of HT3-PSII, HT3-∆Q-PSII, and HT3-∆O-PSII…………...….…………………………………………………………….161 Figure 6 Isolation of two kinds of PSII complexes from the Q-His strain of Synechocystis 6803………...….…...…………………………………………….163 Figure 7 Polypeptide compositions of Complex 1 and Complex 2……………………….164 Figure 8 Two-dimensional Blue-Native-PAGE and immunoblot analysis of Complex 1 and Complex 2………………………………………………….………………...……..166 Figure 9 Relative quantification of the PsbQ protein in Complex 1 and Complex 2…………......168 Figure 10 Schematic model for the binding of PsbQ to the interface of dimeric PSII………..……170 Figure 11 A schematic model for PSII assembly and repair………………………………………..177 Figure S1 Phylogenetic tree showing evolutionary relationship between PsbQ sequences from different cyanobacterial species…………… …..….…………………...…………188 Figure S2 BS3-induced cross-links between PsbQ and PsbO determined by tandem LC-MS and a subsequent database search using MassMatrix…......…………………....189 Figure S3 Mass spectrometric data showing an intermolecular PsbQ-PsbQ cross-linked peptide...190 Figure S4 Structural and electrostatic-potential comparison of PsbO from Synechocystis 6803 and T. ulcanus……......……………………...…………………......191 Figure S5 EDC-induced cross-links between PsbQ and CP47 determined by tandem LC-MS/MS and a subsequent database search in MassMatrix…..……………..…...…..192 vi Figure S6 Distance between K96 and K120 in the Synechocystis 6803 PsbQ crystal structure…...193 Chapter 5: Conclusions, future directions, and additional work Figure 1 Glycerol gradient ultracentrifugation of purified PSII complexes reveals an unidentified subcomplex in the ΔpsbO-His47-PSII sample………….……........202