Washington University in St. Louis Washington University Open Scholarship Arts & Sciences Electronic Theses and Dissertations Arts & Sciences Spring 5-15-2017 Mass Spectrometry-Based Structural Analysis of Photosynthetic Protein Assemblies Yue Lu Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/art_sci_etds Part of the Biochemistry Commons Recommended Citation Lu, Yue, "Mass Spectrometry-Based Structural Analysis of Photosynthetic Protein Assemblies" (2017). Arts & Sciences Electronic Theses and Dissertations. 1127. https://openscholarship.wustl.edu/art_sci_etds/1127 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 IN ST. LOUIS Department of Chemistry Dissertation Examination Committee: Robert E. Blankenship, Co-Chair Michael L. Gross, Co-Chair Dewey J. Holten Himadri B. Pakrasi Gary J. Patti Mass Spectrometry-Based Structural Analysis of Photosynthetic Protein Assemblies by Yue Lu A dissertation presented to The Graduate School of Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy May 2017 St. Louis, Missouri © 2017, Yue Lu Table of Contents List of Figures ................................................................................................................................ vi List of Tables .................................................................................................................................. x List of Abbreviations ..................................................................................................................... xi Acknowledgments........................................................................................................................ xiii Abstract ....................................................................................................................................... xiiii Chapter 1: Introduction ................................................................................................................... 1 1.1 Protein machineries involved in photosynthesis .............................................................. 1 1.2 Protein primary structure determination .......................................................................... 3 1.3 MS-based Footprinting ..................................................................................................... 6 1.4 CROSSLINKING (XL)-MS ............................................................................................ 8 1.5 Top-down MS .................................................................................................................. 9 1.6 Challenges in photosynthetic membrane protein MS study ........................................... 11 Chapter 2: Top-down Mass Spectrometry Analysis of Membrane-bound Light-Harvesting Complex 2 from Rhodobacter sphaeroides ................................................................................... 15 2.1 Abstract .......................................................................................................................... 15 2.2 Introduction .................................................................................................................... 16 2.3 Materia and Methods ...................................................................................................... 21 2.3.1 LH2 Preparation .................................................................................................................. 21 2.3.2 Top-down LC-MS analysis of LH2 .................................................................................... 21 2.3.3 puc2B gene identification ................................................................................................... 22 2.3.4 puc2B mRNA identification ............................................................................................... 22 2.3.5 Homology modelling .......................................................................................................... 23 2.4 Results and discussion .................................................................................................... 23 2.4.1 Composition of mature LH2 complexes ........................................................................... 23 2.4.2 Sequence and post translational modifications ................................................................... 25 2.5.1 Substitution of Valine for Isoleucine .................................................................................. 37 2.5 Conclusions .................................................................................................................... 41 ii Chapter 3: Mapping the Topology and Conformation of an Intrinsic Membrane Protein in a Lipid Bilayer by Fast Photochemical Oxidation of Proteins (FPOP) ..................................................... 42 3.1 Abstract .......................................................................................................................... 43 3.2 Introduction .................................................................................................................... 47 3.3 Materials and Methods ................................................................................................... 47 3.3.1 LH2 preparation .................................................................................................................. 47 3.3.2 MSP preparation ................................................................................................................. 47 3.3.3 Self-assembly of Nanodisc .................................................................................................. 47 3.3.4 Steady-state absorption spectroscopy ................................................................................. 48 3.3.5 Time-resolved fluorescency spectroscopy .......................................................................... 48 3.3.6 Dynamic light scattering ..................................................................................................... 49 3.3.7 Footprinting of Nanodisc-LH2 and detergent micelle-embedded LH2 .............................. 49 3.3.8 MS analysis ......................................................................................................................... 50 3.3.9 Data analysis ....................................................................................................................... 51 3.3.10 Sequence alignment, topology prediction and homology modeling ................................... 51 3.4 Results and discussion .................................................................................................... 51 3.4.1 Characterization of the Nanodisc containing LH2 ............................................................ 51 3.4.2 Application of FPOP to membrane proteins ....................................................................... 54 3.4.3 Membrane protein in detergent micelle vs. Nanodisc ......................................................... 56 3.4.4 Methionine as a marker in membrane protein labeling by FPOP ..................................... 61 3.4.5 Locating the membrane protein in the lipid bilayer ............................................................ 62 3.5 Conclusions .................................................................................................................... 74 Chapter 4: Mass Spectrometry Characterization of Reaction Center from Blastochloris viridis— —the First Integral Membrane Protein Complex Determined by X-ray Crystallography ........... 75 4.1 Abstract .......................................................................................................................... 75 4.2 Introduction .................................................................................................................... 76 4.3 Materials and Methods ................................................................................................... 78 4.3.1 Cell culture and reaction center preparation ....................................................................... 78 4.3.2 Top-down LC-MS ............................................................................................................... 78 4.3.3 Bottom-up LC-MS .............................................................................................................. 79 4.3.4 Native MS analysis of reactions center ............................................................................... 80 4.3.5 MS data processing ............................................................................................................. 80 iii 4.4 Results and discussion .................................................................................................... 81 4.4.1 Top-down MS analysis of denatured reaction center ........................................................ 81 4.4.2 Bottom-up MS analysis of reaction center .......................................................................... 82 4.4.3 Native MS analysis of reaction center ................................................................................ 89 4.5 Conclusions ...................................................................................................................