UC Irvine UC Irvine Electronic Theses and Dissertations Title Investigating Protein Complex Dynamics: Analysis of Cullin-Ring Ligase Machinery through Development of Quantitative Cross-linking Mass Spectrometry Strategies Permalink https://escholarship.org/uc/item/6164k1z6 Author Yu, Clinton Publication Date 2017 Supplemental Material https://escholarship.org/uc/item/6164k1z6#supplemental Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, IRVINE Investigating Protein Complex Dynamics: Analysis of Cullin-Ring Ligase Machinery through Development of Quantitative Cross-linking Mass Spectrometry Strategies DISSERTATION Submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILOSOPHY In Biological Sciences By Clinton Yu Dissertation Committee: Professor Lan Huang, Chair Professor Todd Holmes Professor Rongsheng Jin Professor Feng Qiao 2017 TABLE OF CONTENTS LIST OF FIGURES ...................................................................................................................... v LIST OF TABLES ...................................................................................................................... vii ACRONYMS & SYMBOLS ..................................................................................................... viii ACKNOWLEDGMENTS ........................................................................................................... ix CURRICULUM VITAE ............................................................................................................... x ABSTRACT OF THE DISSERTATION ................................................................................ xiv CHAPTER 1: Introduction .......................................................................................................... 1 1.1 Significance of Protein-Protein Interactions ............................................................... 1 1.1.1 Traditional Methods for PPI Discovery .............................................................................. 2 1.1.2 Traditional Methods for Structural Elucidation .................................................................. 3 1.2 Cross-linking Mass Spectrometry as a Tool for Studying PPIs ................................ 4 1.2.1 Chemical Cross-linking Coupled with Affinity-Purification Mass Spectrometry .............. 5 1.2.2 Chemical Cross-linking as a Hybrid Structural Methodology ............................................ 6 1.2.3 Challenges in Cross-linking Mass Spectrometry Studies ................................................... 7 1.3 The Ubiquitin-Proteasome System ............................................................................... 9 1.4 Cullin-Ring E3 Ubiquitin Ligases (CRLs) ................................................................. 13 CHAPTER 2: Development of Isotope-coded, MS-cleavable Cross-linkers for Elucidating Protein Structures through Quantitative XL-MS .................................................................... 15 2.1 Summary ....................................................................................................................... 15 2.2 Introduction .................................................................................................................. 16 2.3 Experimental Procedures ............................................................................................ 19 2.3.1 Materials and Reagents ..................................................................................................... 19 2.3.2 Synthesis and Characterization of d0-DMDSSO and d10-DMDSSO ................................ 19 2.3.3 Cross-linking of Synthetic Peptides with d0- and d10-DMDSSO. ..................................... 20 2.3.4 Cross-linking of Cytochrome C with d0- and d10-DMDSSO ............................................ 20 2.3.5 Liquid Chromatography-Multistage Tandem Mass Spectrometry (LC-MSn) .................. 21 2.3.6 Data Analysis of Cross-linked Peptides ............................................................................ 21 2.4 Results & Discussion .................................................................................................... 22 2.4.1 Design and Synthesis of New Isotope-coded DSSO Derivatives ..................................... 22 2.4.2 Expected CID Fragmentation Patterns of d0- and d10-DMDSSO Cross-linked Peptides .. 23 2.4.3 Characterization of DMDSSO Cross-linked Model Peptides by MSn Analysis ............... 25 ii n 2.4.4 Characterization of DMDSSO Cross-linked Cytochrome C by MS Analysis ................ 27 2.4.5 Detection of d0/d10-DMDSSO Cross-linked Peptide Pairs ............................................... 30 2.4.6 Quantitation of d0-/d10-DMDSSO Cross-linked Peptides ................................................. 33 2.5 Conclusion .................................................................................................................... 35 CHAPTER 3: Gln40 deamidation Blocks Structural Reconfiguration and Activation of SCF Ubiquitin Ligase Complex by Nedd8 ................................................................................ 39 3.1 Summary ....................................................................................................................... 39 3.2 Introduction .................................................................................................................. 40 3.3 Experimental Procedures ............................................................................................ 42 3.3.1 Materials and Reagents ..................................................................................................... 42 3.3.2 Preparation of Cul1-Rbx1 Protein Complexes.................................................................. 43 3.3.3 Ubiquitination Assays ....................................................................................................... 44 3.3.4 DMDSSO Cross-linking and Digestion of Cul1-Rbx1 Complexes .................................. 45 3.3.5 Liquid Chromatography-Multistage Tandem Mass Spectrometry (LC MSn) ................... 45 3.3.6 Data Analysis, Identification and Quantification of Cross-linked Peptides ..................... 46 3.4 Results & Discussion .................................................................................................... 47 3.4.1 Reconstitution of SCF E3 Activity with Intact Proteins ................................................... 47 3.4.2 Quantitative XL-MS strategy ............................................................................................ 50 3.4.3 Mapping XL-MS Data to Cul1-Rbx1 Complexes ............................................................ 53 3.4.5 Quantitation of DMDSSO Cross-linked Peptides ............................................................. 57 3.4.6 Comparison of Cul1-Rbx1 and Nedd8~Cul1-Rbx1 complexes........................................ 59 3.4.7 Comparison of Nedd8~ and Nedd8(Q40E)~Cul1-Rbx1 complexes................................. 65 3.5 Conclusion .................................................................................................................... 68 CHAPTER 4: Developing a Multiplexed Quantitative Cross-linking Mass Spectrometry Platform for Comparative Structural Analysis of Protein Complexes .................................. 72 4.1 Summary ....................................................................................................................... 72 4.2 Introduction .................................................................................................................. 73 4.3 Experimental Procedures ............................................................................................ 76 4.3.1 Materials and Reagents ..................................................................................................... 76 4.3.2 DSSO Cross-linking of Cytochrome C ............................................................................. 76 4.3.3 TMT2 labeling of Cross-linked Cytochrome C Peptides .................................................. 77 4.3.3 Liquid Chromatography-Multistage Tandem Mass Spectrometric (LC-MSn) Analysis .. 77 4.3.4 Identification and Quantitation of TMT2 Labeled DSSO Cross-linked Peptides ............. 78 4.4 Results and Discussion ................................................................................................. 79 iii 4.4.1 Development of A New Multiplexed QXL-MS Strategy ................................................. 79 4.4.2 Fragmentation of TMT-labeled, DSSO-cross-linked peptides ......................................... 81 4.4.3 MSn Analysis of TMT-labeled Cytochrome C Cross-linked Peptides .............................. 84 4.5 Conclusion .................................................................................................................... 89 CHAPTER 5: Describing the Molecular Mechanisms of Ubiquitination Inhibition through Targeting of E2 Ubiquitin-Conjugating Enzymes ................................................................... 91 5.1 Summary ....................................................................................................................... 91 5.2 Introduction .................................................................................................................. 92 5.3 Experimental Procedures ............................................................................................ 95 5.3.1