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Reductive Alkylation of Proteins Towards Structural and Biological Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2014 Reductive Alkylation of Proteins Towards Structural and Biological Applications Kevin Jerome Roberson Louisiana State University and Agricultural and Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Part of the Chemistry Commons Recommended Citation Roberson, Kevin Jerome, "Reductive Alkylation of Proteins Towards Structural and Biological Applications" (2014). LSU Doctoral Dissertations. 1267. https://digitalcommons.lsu.edu/gradschool_dissertations/1267 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 Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. REDUCTIVE ALKYLATION OF PROTEINS TOWARDS STRUCTURAL AND BIOLOGICAL APPLICATIONS 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 The Department of Chemistry by Kevin Jerome Roberson B.S., Georgia Southern University, 2007 August 2014 To the pursuit of consciousness…surviving is not enough! ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Megan Macnaughtan, for guidance and support throughout my graduate studies. I would also like to thank my committee members, Dr. Isiah Warner, Dr. Kermit Murray, Dr. William Crowe, and Dr. William Monroe for their advice and support. Thanks to Dr. Fareed Aboul-ela for his support and advice as well. I sincerely thank Dr. Dale Treleaven and Dr. Thomas Weldeghiorghis for their invaluable help and teachings of NMR. I thank the Macnaughtan Research Group for their support and friendship over the years. Octavia “Buddarris” Goodwin, you have been one of my biggest supporters in a place so far away from family. I thank you and Mrs. Judy for being my family when I couldn’t reach mine. I give a special thank Dr. Karen Terry Welch and Marion Welch. I am blessed to have crossed paths with you. I thank you for being selfless enough to invest in the potential I couldn’t see in myself. Although these words do no justice to my gratitude, I offer them in a lifelong attempt to fully express how much you mean to me and my family. Just know I’m working on something big… I thank my mom, Diane Roberson, for the many prayers and sacrifices you have made on my behalf so that I may be in this position. My siblings for your unwavering support. Fred, I still want to be like you when I grow up. Michelle, the bond we share has always been special and I’m a much better person because of you. My angels, Jessica and Vanessa, I was always jealous cause I didn’t have a twin. I wouldn’t work and push as hard as I do in life if not for you. This would not be possible without any of you. I love you! Last but definitely not least, I thank Dr. Marsha Cole. Your heart is as pure as they come. I thank you for being there for me throughout this journey. Your support always seem to grow iii when it was hardest to be there for me. Thank you for sacrificing sleep with me, challenging me, and somehow always polishing any idea I presented to you. It did not go unnoticed. I thank you for helping me achieve such a milestone in my life, and I look forward to the amazing things you will produce in the future. I love you! -Kevin J. Roberson iv TABLE OF CONTENTS ACKNOWLEDGEMENTS ........................................................................................................... iii LIST OF TABLES ....................................................................................................................... viii LIST OF SCHEMES...................................................................................................................... ix LIST OF FIGURES .........................................................................................................................x ABSTRACT ............................................................................................................................... xii CHAPTER 1 INTRODUCTION .....................................................................................................1 1.1 Protein Structure and Stability .................................................................................1 1.1.1 Protein Structure ................................................................................................1 1.1.2 Classification of Protein Structure .....................................................................6 1.1.3 Protein Denaturation ..........................................................................................8 1.2 Protein Modification ..............................................................................................11 1.2.1 Protein Synthesis .............................................................................................12 1.2.2 Post-translational Modification .......................................................................13 1.2.3 Recombinant Protein Expression .....................................................................15 1.2.4 Chemical Modification ....................................................................................17 1.2.5 Side Chain Selective Modifications.................................................................19 1.3 Reductive Methylation of Proteins ........................................................................26 1.3.1 Techniques to Study Reductive Methylation of Proteins ................................28 1.3.2 Protein Structure: NMR and X-Ray Crystallography ......................................30 1.3.3 Protein Samples for NMR ...............................................................................34 1.3.4 NMR Assignment Strategies ...........................................................................36 1.3.5 Assignment of Reductively Methylated HEWL ..............................................42 1.4 Conclusion .............................................................................................................48 1.5 References ..............................................................................................................48 CHAPTER 2 ATTEMPTS TOWARD UNAMBIGUOUSLY ASSIGNING 13C- DIMETHYLAMINE NMR RESONANCES ..........................................................59 2.1 Introduction ............................................................................................................59 2.2 Experimental ..........................................................................................................61 2.2.1 Reductive Methylation in the Presence of 18C6 .............................................61 2.2.2 Fully 13C-labeled Control Sample ...................................................................61 2.2.3 Partial 13C-labeling ..........................................................................................62 2.2.4 Excess Natural Abundance Formaldehyde to Complete Methylation .............62 2.2.5 Reductive Methylation Using Multiple Reducing Agents ...............................62 2.2.6 Preparation for NMR .......................................................................................63 v 2.2.7 NMR ................................................................................................................63 2.2.8 13C Percent Incorporation Calculation from NMR Data .................................63 2.2.9 Non-destructive Edman degradation ...............................................................64 2.3 Results and Discussion ..........................................................................................64 2.3.1 Reductive Methylation in the Presence of 18-Crown-6-Ether ........................64 2.3.2 Reductive Methylation Using Multiple Reducing Agents ...............................67 2.3.3 Non-destructive Edman Degradation ..............................................................69 2.4 Conclusion .............................................................................................................71 2.5 References ..............................................................................................................71 CHAPTER 3 METHOD TO IDENTIFY THE NMR RESONANCES OF THE 13C-DIMETHYL N-TERMINAL LYSINE ON REDUCTIVELY METHYLATED PROTEIN .......................................................75 3.1 Introduction ............................................................................................................75 3.2 Experimental ..........................................................................................................78 3.2.1 Materials and Methods ....................................................................................78 3.2.2 Reductive Methylation Lysozyme (Generic Procedure) .................................78 3.2.3 MALDI-TOF Sample Preparation ...................................................................79 3.2.4 MALDI-TOF Analysis ....................................................................................80 3.2.5 Determination of the Percent 13C Labelling at Each Site: NMR .....................80 3.2.6 Mass Spectrometry ..........................................................................................81 3.3 Results ....................................................................................................................82
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