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Genetically Engineered Aequorin for the Development of Novel Bioanalytical Systems University of Kentucky UKnowledge University of Kentucky Doctoral Dissertations Graduate School 2011 GENETICALLY ENGINEERED AEQUORIN FOR THE DEVELOPMENT OF NOVEL BIOANALYTICAL SYSTEMS Krystal Teasley Hamorksy University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Hamorksy, Krystal Teasley, "GENETICALLY ENGINEERED AEQUORIN FOR THE DEVELOPMENT OF NOVEL BIOANALYTICAL SYSTEMS" (2011). University of Kentucky Doctoral Dissertations. 182. https://uknowledge.uky.edu/gradschool_diss/182 This Dissertation is brought to you for free and open access by the Graduate School at UKnowledge. It has been accepted for inclusion in University of Kentucky Doctoral Dissertations by an authorized administrator of UKnowledge. For more information, please contact [email protected]. ABSTRACT OF DISSERTATION Krystal Teasley Hamorsky The Graduate School University of Kentucky 2011 GENETICALLY ENGINEERED AEQUORIN FOR THE DEVELOPMENT OF NOVEL BIOANALYTICAL SYSTEMS ABSTRACT OF DISSERTATION A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Arts and Sciences at the University of Kentucky By Krystal Teasley Hamorsky Lexington, Kentucky Director: Dr. Sylvia Daunert, Professor of Chemistry Lexington, Kentucky 2011 Copyright © Krystal Teasley Hamorsky 2011 ABSTRACT OF DISSERTATION GENETICALLY ENGINEERED AEQUORIN FOR THE DEVELOPMENT OF NOVEL BIOANALYTICAL SYSTEMS The ability to rationally or randomly modify proteins has expanded their employment in various bioanalytical applications. The bioluminescent protein, aequorin, has been employed as a reporter for decades due to its simplistic, non-hazardous nature and its high sensitivity of detection. More recently aequorin has been subject to spectral tuning. Techniques such as random and site-directed mutagenesis, the incorporation of coelenterazine analogues and the incorporation of non-natural amino acids have expanded the palette of aequorin by altering their emission wavelengths and/or half-lifes. Due to the increased diversity of aequorin, it can be used in multianalyte detection. Although aequorin has been studied extensively and has been used as a reporter in a wide array of applications, it has never been employed as a reporter in systems that involve the splitting of aequorin. Herein we describe the splitting of aequorin in such a way where it becomes the reporter protein in the development of protein-based molecular switches. We have created two distinct protein switches by genetically inserting the glucose-binding protein and the sulfate-binding protein into the aequorin sequence, splitting it in such a manner that it allows for the selective detection of glucose and sulfate, respectively. In a separate investigation, we developed a bioluminescence inhibition binding assay for the detection of hydroxylated polychlorinated biphenyls. These systems have shown that they can be employed in the detection of the respective analyte in biological as well as in environmental samples, which demonstrated a sensitive, fast alternative approach to current methods for on-site screening. Furthermore, we propose the rational design, preparation and use of truncated aequorin fragments in bioanalytical platforms such as multi-analyte detection, protein complementation assays and protein tagging assays based on our discovery that truncated aequorin retains partial bioluminescence emission. One such truncated aequorin demonstrated a large red shift in the emission maximum. It is envisioned that this new red-shifted truncated aequorin will find applications in multi-analyte detection. We anticipate that this work will lead to the discovery of additional functional truncated aequorin fragments that can be employed in novel protein-protein interactions or protein folding systems. KEYWORDS: Photoprotein, Molecular Switch, Inhibition Assays, Binding Proteins, Truncated Proteins Krystal Teasley Hamorsky April, 23 2011 GENETICALLY ENGINEERED AEQUORIN FOR THE DEVELOPMENT OF NOVEL BIOANALYTICAL SYSTEMS By Krystal Teasley Hamorsky Sylvia Daunert Director of Dissertation John Anthony Director of Graduate Studies April, 23 2011 RULES FOR THE USE OF DISSERTATIONS Unpublished dissertations submitted for the Doctor’s degree and deposited in the University of Kentucky Library are as a rule open for inspection, but are to be used only with due regard to the rights of the authors. Bibliographical references may be noted, but quotations and summaries of parts may be published only with the permission of the author, and with the usual scholarly acknowledgments. Extensive copying or publication of the dissertation in while or in part also requires the consent of the Dean of the Graduate School of the University of Kentucky. A library that borrows this dissertation for use by its patrons is expected to secure the signature of each user. Name Date DISSERTATION Krystal Teasley Hamorsky The Graduate School University of Kentucky 2011 GENETICALLY ENGINEERED AEQUORIN FOR THE DEVELOPMENT OF NOVEL BIOANALYTICAL SYSTEMS DISSERTATION A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Arts and Sciences at the University of Kentucky By Krystal Teasley Hamorsky Lexington, Kentucky Director: Dr. Sylvia Daunert, Professor of Chemistry Lexington, Kentucky 2011 Copyright © Krystal Teasley Hamorsky 2011 For my nieces, Kallie, Jersey and Amaya ACKNOWLEDGMENTS This dissertation would not have been made possible without the support of several people. First and foremost, I would like to thank my advisor, Dr. Sylvia Daunert, for her outstanding encouragement and guidance throughout my graduate career. She has allowed me to working independently, supported my ideas and given me a great deal of assistance along the way. Without her, my dissertation would not be possible. I am particularly grateful to her for the funding she has provides me with grants from the National Institutes of Health and the National Institutes of Environmental Health. I am also thankful for a Superfund Research Fellowship as well as Gill Fellowship. Furthermore, I would like to thank my committee members, Dr. Leonidas Bachas, Dr. Kim Anderson, and Dr Mark Lovell, for their time and effort throughout my research. Special thanks go to Dr. Bachas for his helpful advice and suggestions during group meetings. I am forever grateful for Dr. Mark Ensor, who has been amazing in guiding me through molecular biology. He deserves many thanks for teaching me countless techniques in addition to editing my papers and dissertation. Moreover, he has been encouraging and I have enjoyed our early morning talks. Dr. Emre Dikici also deserves gratitude for all of our conversations about my research leading to many helpful suggestions. He also merits acknowledgement for revising my dissertation and assistance in making figures in chapters one and two. Additionally, I would like to thank Dr. Yinan Wei for aid in chapter two. She trained me to use the circular dichroism instrument which was influential to chapter two’s publication. Furthermore she assisted in interpretation of iii the data. Dr. Patrizia Pazini deserves thanks for her analytical insight in chapters three and four and I appreciate her help in revising all chapters of my disseration. I am very thankful for the group members, past and present, of Dr. Daunert’s and Dr. Bachas’ research groups. I am grateful for the scientific talks that I have share with them, especially Leslie Doleman Knecht and Kendrick Turner whom I trouble shoot with on regular basis. I have enjoyed getting to know the lab members and I am thankful for the friendships that have developed. I am especially grateful for my family for their endless love and support. Even though most of my family does not understand my research or anything about graduate school they have always been there when I have needed them. I want to thank my mom for being my best friend and for all of our phone conversation that have helped me tackle life. I also want to thank my grandparents for always being there for me and the encouragement they have given me. Moreover, I appreciate my nieces for being wonderful and keeping me motivated when my motivation seems lost. They do not know how special they are to me. Last but not least, I indebted to my husband, Kyle for his patience, love and support. He has stood by me through thick and thin, and I would not have made it without him. He has been there to encourage me and lift me up when I am down, research or life. I am thankful for all the stressful moments that he has made better. He has helped me keep my motivation and celebrate each successful step of my career. iv TABLE OF CONTENTS Acknowledgments.............................................................................................................. iii List of Tables .................................................................................................................... vii List of Figures .................................................................................................................. viii Chapter One: Introduction Bioluminescence ......................................................................................................1 Photoproteins ...........................................................................................................2 Aequorin ..................................................................................................................3
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