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Using Electron Microscopy to Gain Structural Insight Into USING ELECTRON MICROSCOPY TO GAIN STRUCTURAL INSIGHT INTO BIOLOGICALLY RELEVANT, LABILE OR DESTABILIZED PROTEIN COMPLEXES by HARRY WILLIAM SCOTT III Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Advisor: Dr. Derek J. Taylor Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY January 2019 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of Harry William Scott III Candidate for the Doctor of Philosophy degree *. (Thesis Advisor) Derek Taylor Ph.D. (Committee Chair) Jason Mears Ph.D. (Committee Member) Masaru Miyagi Ph.D. (Committee Member) Eckhard Jankowsky Ph.D. (Committee Member) Phoebe Stewart Ph.D. Date of Defense 11/19/18 * We also certify that written approval has been obtained for any proprietary material contained therein ii To Aiman and Yasmeen. iii TABLE OF CONTENTS LIST OF TABLES ........................................................................................................... viii LIST OF FIGURES ........................................................................................................... ix LIST OF ABBREVIATIONS ............................................................................................ xi ACKNOWLEDGEMENTS ............................................................................................. xvi ABSTRACT ........................................................................................................................ 1 CHAPTER 1 : INTRODUCTION AND BACKGROUND ............................................... 3 1.1 Why do we use electron microscopy? .................................................................. 4 1.2 The structure of the S. pombe telomere ............................................................... 5 1.2.1 Shelterin-Telomerase interplay at the S. pombe Telomere .................................. 6 1.2.2 Roles of individual shelterin components and mammalian homologues ............. 7 1.2.3 Double-stranded telomeric DNA binding shelterin proteins................................ 9 1.2.4 Single-stranded telomeric DNA binding shelterin proteins ............................... 11 1.2.5 Shelterin proteins that do not bind DNA ............................................................ 13 1.2.6 The importance of the S. pombe shelterin Ccq1-Tpz1-Poz1 subcomplex ......... 18 1.3 Anthrax toxin delivery ............................................................................................ 22 1.3.1 The protective antigen protein .............................................................................. 23 1.3.2 Pore-formation in the PA heptamer ..................................................................... 24 1.3.3 Passage of lethal factor and edema factor ............................................................ 26 1.3.4 Mutants that abolish pore-forming activity .......................................................... 30 iv 1.3.5 Nodes of inhibition ............................................................................................... 32 1.4 Translation of these results to other β-pore forming proteins – adapted from review (Scott et al. 2018) .......................................................................................................... 34 1.5 How EM can bridge structural gaps ........................................................................ 38 1.5.1 Sample preparation ............................................................................................... 40 1.5.2 Structure determination using EM ....................................................................... 40 1.5.3 Image processing .................................................................................................. 42 1.5.4 Sorting Structural Heterogeneity in Protein Complexes ...................................... 47 CHAPTER 2 : SPATIAL ORGANIZATION AND MOLECULAR INTERACTIONS OF THE SCHIZOSACCAROMYCES POMBE CCQ1-TPZ1-POZ1 SHELTERIN COMPLEX ....................................................................................................................... 58 2.1 ABSTRACT ............................................................................................................ 59 2.2 INTRODUCTION ................................................................................................... 60 2.3 RESULTS ................................................................................................................ 63 2.4 DISCUSSION ......................................................................................................... 68 2.5 ACCESSION NUMBERS ...................................................................................... 71 2.6 AUTHOR CONTRIBUTIONS ............................................................................... 71 2.7 ACKNOWLEDGEMENTS .................................................................................... 71 v CHAPTER 3 : STRUCTURAL CHARACTERIZATION OF THE ANTHRAX PORE INTERMEDIATE SHEDS LIGHT ON THE MECHANISM OF PH-INDUCED, MEMBRANE-SPANNING PORE MATURATION ....................................................... 98 3.1 ABSTRACT ............................................................................................................ 99 3.2 INTRODUCTION ................................................................................................. 100 3.3 RESULTS .............................................................................................................. 101 3.4 DISCUSSION ....................................................................................................... 106 3.5 MATERIALS AND METHODS .......................................................................... 109 3.6 ACCESSION NUMBERS .................................................................................... 115 3.7 AUTHOR CONTRIBUTIONS ............................................................................. 115 3.8 ACKNOWLEDGEMENTS .................................................................................. 115 CHAPTER 4 : SUMMARY OF DISCOVERIES AND FUTURE DIRECTIONS ....... 130 4.1 Structural analysis of labile and destabilized protein complexes .......................... 131 4.2 The CTP complex, telomeres and cancer .............................................................. 131 4.3 Shelterin components likely act as dimers ............................................................ 133 4.4 The anthrax D425A PA mutant forms a stable intermediate-pore complex at low pH ................................................................................................................................ 136 4.5 Cryo-EM to answer prevailing questions in the anthrax field............................... 139 4.7 Intermediate pore structures can potentially be used for drug development ......... 143 4.8 Concluding remarks .............................................................................................. 144 vi REFERENCES ............................................................................................................... 146 vii LIST OF TABLES Table 1. Effects of amino acid substitutions in 2β7-2β8 and 2β10-2β11 loops of domain 2 on various PA functions. ................................................................................................... 55 Table 2. Characterization of dominant-negative mutations in PA. ................................... 56 Table 3. List of β-PFPs from recent cryo-EM studies revealing the architecture of prepore/pore assemblies. ................................................................................................... 57 Table 4. Statistics and model refinement for the Anthrax pore-intermediate ................. 129 viii LIST OF FIGURES Figure 1.1. Organization of the Mammalian and S. pombe Shelterin Complexes ........... 50 Figure 1.2. Crystal Structures of moieties associated with shelterin complexes .............. 52 Figure 1.3. Mechanism of Action for Anthrax Pore Formation and Toxin Delivery ....... 53 Figure 1.4. Mechanism of Action for Anthrax Pore Formation and Toxin Delivery ....... 54 Figure 2.1. Schematic representation depicting the diverse roles of the CTP complex in telomere homeostasis. ....................................................................................................... 73 Figure 2.2. The three-dimensional structure of the CTP complex reveals a cage-like complex assembled as a dimer of heterotrimers. .............................................................. 75 Figure 2.3. Localization of the individual protein subunits in the CTP complex. ............ 78 Figure 2.4. MSn analysis of an inter-subunit DSSO cross-link between Ccq1 and Poz1 peptides. ............................................................................................................................ 80 Figure 2.5. Comparison of the shelterin complex subunit organization in S. pombe and in mammals. .......................................................................................................................... 82 Figure 2.6. RCT reconstruction of the CfTP complex. ..................................................... 83 Figure 2.7. Resolution of the CfTP complex and particles used for CfTP, CnTP, mCfTP and CfTmP ......................................................................................................................... 84 Figure 2.8. Biochemical and EM analysis of the CnTP complex...................................... 85 Figure 2.9. Biochemical and
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