University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2014 Allostery and Applications of the Lac Repressor Matthew Almond Sochor University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biochemistry Commons, and the Biophysics Commons Recommended Citation Sochor, Matthew Almond, "Allostery and Applications of the Lac Repressor" (2014). Publicly Accessible Penn Dissertations. 1448. https://repository.upenn.edu/edissertations/1448 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/1448 For more information, please contact [email protected]. Allostery and Applications of the Lac Repressor Abstract The lac repressor has been extensively studied for nearly half a century; this long and complicated experimental history leaves many subtle connections unexplored. This thesis sought to forge those connections from isolated and purified components up ot functioning lac genetic switches in cells and even organisms. We first connected the genetics and structure of the lac repressor to in vivo gene regulation in Escherichia coli. We found that point mutations of amino acids that structurally make specific contacts with DNA can alter repressor-operator DNA affinity and even the conformational equilibrium of the repressor. We then found that point mutations of amino acids that structurally make specific contacts with effector molecules can alter repressor-effector affinity and the conformational equilibrium. Allesults r are well explained by a Monod, Wyman, and Changeux model of allostery. We next connected purified in vitro components with in vivo gene regulation in E. coli. We used an in vitro transcription assay to measure repressor-operator DNA binding affinity, repressor-effector binding affinity, and conformational equilibrium. Only the repressor-operator DNA binding affinity disagreed with literature values from other in vitro experiments, however it did agree with a published value which should hold under in vivo conditions. We were able to use our in vitro thermodynamic parameters to accurately predict the in vivo gene regulation when cell crowding was considered. Finally we developed an autogenously regulated lac repressor for AAV-mediated gene therapy. We were able to improve the gene regulation of the autogenous switch by using multiple operator DNA sites, a tetrameric lac repressor, and point mutations to the lac repressor. The autogenous switch was shown to function in various cell types and was capable of reversible regulation of luciferase in living mice. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Biochemistry & Molecular Biophysics First Advisor Mitchell Lewis Keywords allostery, autogenous switch, gene therapy, lac repressor Subject Categories Biochemistry | Biophysics This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/1448 ALLOSTERY AND APPLICATIONS OF THE LAC REPRESSOR Matthew Almond Sochor A DISSERTATION in Biochemistry and Molecular Biophysics Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2014 Supervisor of Dissertation _________________________ Mitchell Lewis, D.Phil, John Morgan Professor of Biomedical Research and Education Graduate Group Chairperson _________________________ Kathryn M. Ferguson, Ph.D., Associate Professor of Physiology Dissertation Committee: Kim A. Sharp, Ph.D., Associate Professor of Biochemistry and Biophysics Gregory D. Van Duyne, Ph.D., Professor of Biochemistry and Biophysics Kristen W. Lynch, Ph.D., Associate Professor of Biochemistry and Biophysics Mark Goulian, Ph.D., Professor of Biology James Shorter, Ph.D., Associate Professor of Biochemistry and Biophysics Robert Fairman, Ph.D., Professor of Biology, Haverford College ALLOSTERY AND APPLICATIONS OF THE LAC REPRESSOR COPYRIGHT 2014 Matthew Almond Sochor This work is licensed under the Creative Commons Attribution- NonCommercial-ShareAlike 3.0 License To view a copy of this license, visit http://creativecommons.org/licenses/by-ny-sa/2.0/ Dedication To my grandmother Ruth Sochor. She raised three successful kids on her own and made the opportunities I’ve had possible. Upon being asked how it feels to be a great grandmother, she replied, “I’ve always been great. Now you just have to admit it.” iii Acknowledgements I’d like to thank my parents for putting up with me throughout the years and generally being wonderful and supportive people. Also, thanks to Bill for being a good older brother and keeping me in my place. I’ve met too many characters in the greater Philadelphia area during my time here that have impacted my life in many ways, both positive and negative. Thanks for challenging me to learn new things, from playing the accordion to protein purification, and generally making my 20s a good time. Special thanks to the MRI guys. The drinks, quizzo, and political arguments kept me sane. A huge thank you to the the various scientists I have worked with over the years. Bob Daber and Leslie Milk both were excellent lab mates and helped get me started. My classmates have provided endless discussion and support over the years, special thanks to Ed Ballister, Josh Allen, Devin Dersh, and Morgan DeSantis. Finally, my collaborators in the Bennett lab made Chapter 5 possible. Special thanks to Adam Wojno, Theodore Drivas, Latha Vasireddy, Jeannette Bennicelli, Daniel Chung and of course Jean Bennett. Thank you to my beautiful wife Elizabeth, you know I couldn’t have even come close to finishing this without both your support and your brain. Finally, I’d like to acknowledge my son Iggy. You have given me perspective on what is important in life. iv ABSTRACT ALLOSTERY AND APPLICATIONS OF THE LAC REPRESSOR Matthew Almond Sochor Mitchell Lewis The lac repressor has been extensively studied for nearly half a century; this long and complicated experimental history leaves many subtle connections unexplored. This thesis sought to forge those connections from isolated and purified components up to functioning lac genetic switches in cells and even organisms. We first connected the genetics and structure of the lac repressor to in vivo gene regulation in Escherichia coli. We found that point mutations of amino acids that structurally make specific contacts with DNA can alter repressor-operator DNA affinity and even the conformational equilibrium of the repressor. We then found that point mutations of amino acids that structurally make specific contacts with effector molecules can alter repressor-effector affinity and the conformational equilibrium. All results are well explained by a Monod, Wyman, and Changeux model of allostery. We next connected purified in vitro components with in vivo gene regulation in E. coli. We used an in vitro transcription assay to measure repressor-operator DNA binding affinity, repressor-effector binding affinity, and conformational equilibrium. Only the repressor-operator DNA binding affinity disagreed with literature values from other in vitro experiments, however it did agree with a published value which should hold under in vivo conditions. We were able to use our in vitro thermodynamic parameters to accurately predict the in vivo gene regulation when cell crowding was considered. Finally we developed an autogenously regulated lac repressor for AAV-mediated gene therapy. We were able to improve the gene regulation of the autogenous switch by using multiple operator DNA sites, a tetrameric lac v repressor, and point mutations to the lac repressor. The autogenous switch was shown to function in various cell types and was capable of reversible regulation of luciferase in living mice. vi Table of Contents Dedication......................................................................................................................iii Acknowledgements.......................................................................................................iv Abstract..................................................................Error: Reference source not found Table of Contents.........................................................................................................vii List of Tables..................................................................................................................x List of Illustrations........................................................................................................xi Chapter 1 – The Lac Repressor 1.1 Introduction.............................................................................................1 1.2 Structure and Function in the Lac Operon...........................................3 1.3 Protein Structure and the Boltzmann..................................................11 1.4 Monod, Wyman, and Changeux (MWC) Model of Allostery...............15 1.5 The MWC Model of Lac Repressor Genetic Regulation.....................24 1.6 Mutations to the Lac Repressor Alters Gene Regulation...................26 1.7 Aims.......................................................................................................28 Chapter 2 – Materials and Methods 2.1 Bacterial Strains and Media.................................................................31 2.2 Eukaryotic Cell Lines and Media.........................................................32 2.3 Plasmid Preparation.............................................................................33