Louisiana State University LSU Digital Commons LSU Historical Dissertations and Theses Graduate School 1993 The iS gnificance of Beta-72 Asparagine Methylation in C-Phycocyanin. Beth Ann Thomas Louisiana State University and Agricultural & Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_disstheses Recommended Citation Thomas, Beth Ann, "The iS gnificance of Beta-72 Asparagine Methylation in C-Phycocyanin." (1993). LSU Historical Dissertations and Theses. 5676. https://digitalcommons.lsu.edu/gradschool_disstheses/5676 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. For more information, please contact [email protected]. INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. 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Ann Arbor, Ml 48106 THE SIGNIFICANCE OF BETA-72 ASPARAGINE METHYLATION IN C-PHYCOCYANIN A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Department of Biochemistry by Beth Ann Thomas B.S., Louisiana State University, 1988 December 1993 Acknowledgements I would like to thank my thesis advisor, Professor Alan Klotz, for his learned and astute guidance in the laboratory. He never ceased to believe in me and my abilities and very patiently taught me the workings of biochemistiy and in particular, protein chemistry. I owe him a great deal of gratitude for his patience and understanding, and his neverending and delightful sense of humor. I am also greatly indebted to Professor Jeff Nelson, who patiently taught me NMR spectroscopy and molecular mechanics/dynamics methodologies (even at 4 in the afternoon). It is obvious from the results in this document that Jeff provided a great deal of assistance with CHARMM and manipulation of NMR pulse routines. I also greatly appreciate the assistance of Professor Terry Bricker in the photosynthetic characterizations of the methylase-minus mutants; I value his enthusiasm for my thesis project. I would also like to thank the other members of my committee who were valuable consultants: Professor George Stanley, Professor Simon Chang and Professor Ezzat Younathan. I have many wonderful friends at Chapel of the Cross Lutheran Church including Virginia Simoneaux, Dick Felder, Mike Lewis, John Roth, Gary Peterson, Audra Gossett and many others; I thank them for the continual interest in my project and the enormous amounts of emotional and spiritual support. Finally, I thank my parents for their constant financial support throughout graduate school and for the home-cooked meals provided to me during the last few months of graduate school. Table of Contents Acknowledgements .............................................................................................. ii List of Tables .................................................................................................... vi List of Figures ................................................................................................ viii Abbreviations and Trivial Names ................................................................. x A bstract................................................................................................................. xii Chapter 1. Introduction ................................................................................. 1 I. Post-Translational Protein Modifications ....................................... 1 II. Light-Harvesting/Light Reactions of Photosynthesis ...................... 7 III. Light-Harvesting of Antennae of Cyanobacteria ......................... 11 IV. NMA and its Relationship to C-Phycocyanin ............................... 18 V. Scope of Thesis Research ........................................................... 29 Chapter 2. Detection of Methylated Asparagine and Glutamine Residues in Polypeptides ............................................................. 32 I. Introduction ..................................................................................... 32 II. Materials and Methods ................................................................ 33 A. Chemicals............................................................................... 33 B. Sequencing of Peptides containing y-N-Methylasparagine 33 C. Preparation of O- and N-Methyl Amino Acid Derivatives 34 D. Acid Hydrolysis ................................................................... 35 E. Amino Acid Analysis ............................................................. 35 F. Preparation of PTH Derivatives ........................................... 35 G. NMR Analysis ................................... 36 H. Mass Spectrometry ............................................................... 36 III. Results and Discussion ............................................................ 37 Chapter 3. Post-Translational Methylation of Phycobilisomes and Oxygen Evolution Efficiency in Cyanobacteria ....................... 51 I. Introduction ................................... 51 II. Materials and Methods ............................................................... 53 A. Strains and Growth Conditions ............................................ 53 B. Oxygen Evolution Measurements ......................................... 54 C. Statistical Analysis of Quantum Yield Data ........................... 55 D. Stoichiometry od Asparagine Methylation .............................. 56 E. Growth Competition Experiments ......................................... 57 III. Results and Discussion ............................................................ 59 A. Asparagine Methylation ........................................................ 61 B. Effect of Asparagine Methylation on Photosynthetic Rates ..................................................................... 63 C. Growth Competition Experiments ......................................... 72 Chapter 4. Characterization of p-72 Site-Specific Mutants in C-Phycocyanin ................................................................................ 82 I. Introduction ...................................................................................... 82 I. M ethods ............................................................................................ 83 A. Mutant Construction ............................................................. 83 B. Strains and Culture Conditions ........................................... 86 C. Phycocyanin Isolation .......................................................... 87 D. Phycobilisome Isolation ....................................................... 88 E. Isoelectric Focusing .............................................................. 89 F. Glutamine Methylation Analysis .......................................... 90 G. Steady-State Oxygen Evolution Experiments ........................ 91 H. Absorbance Spectroscopy ..................................................... 91 I. Fluorescence Spectroscopy .................................................. 92 J. Molecular Mechanics/Dynamics ............................................ 96 II. Results and Discussion ................................................................ 99 A. Characterization of p-72 Mutations ....................................... 99 B. Isoelectric Focusing ............................................................. 103 C. Radiolabel Analysis of Site-Directed Mutants ....................... 108 D. Characterization of Site-Directed Mutants by Oxygen Evolution ............................................................... 115 E. Characterization of Wild-type and Mutant C-Phycocyanins 119 F. Wild-type and Mutant Phycobilisome Characterization 126 G. Time-Resolved