University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Masters Theses Graduate School 12-2009 Applications of BRET: The Detection and Visualization of Protein Interactions and Intramolecular Conformational Changes Lindsay Anne Staron Follow this and additional works at: https://trace.tennessee.edu/utk_gradthes Part of the Biochemistry Commons Recommended Citation Staron, Lindsay Anne, "Applications of BRET: The Detection and Visualization of Protein Interactions and Intramolecular Conformational Changes. " Master's Thesis, University of Tennessee, 2009. https://trace.tennessee.edu/utk_gradthes/560 This Thesis is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Masters Theses by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a thesis written by Lindsay Anne Staron entitled "Applications of BRET: The Detection and Visualization of Protein Interactions and Intramolecular Conformational Changes." I have examined the final electronic copy of this thesis for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Master of Science, with a major in Biochemistry and Cellular and Molecular Biology. Albrecht von Arnim, Major Professor We have read this thesis and recommend its acceptance: Andreas Nebenführ, Elena Shpak, John Biggerstaff Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) To the Graduate Council: I am submitting herewith a thesis written by Lindsay Anne Staron entitled “Applications of BRET: The Detection and Visualization of Protein Interactions and Intramolecular Conformational Changes.” I have examined the final electronic copy of this thesis for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Master of Science, with a major in Biochemistry and Cellular and Molecular Biology. Albrecht von Arnim, Major Professor We have read this thesis and recommend its acceptance: Andreas Nebenführ _______________________ Elena Shpak _______________________ John Biggerstaff _______________________ Accepted for the Council: Carolyn R. Hodges ____________________________ Vice Provost and Dean of the Graduate School (Original signatures are on file with official student records.) Applications of BRET: The Detection and Visualization of Protein Interactions and Intramolecular Conformational Changes A Thesis Presented for the Master of Science Degree The University of Tennessee, Knoxville Lindsay Anne Staron December 2009 Copyright © 2009 by Lindsay Anne Staron All rights reserved. ii ACKNOWLEDGEMENTS There are a number of people whom I wish to thank for their support during my time in the Biochemistry and Cellular, and Molecular Biology department. First and foremost, I would like to thank my mentor, Dr. Albrecht von Arnim, for his continual encouragement and guidance throughout the course of completing my research project. I am very grateful to have had the experiences that came with working in his lab. In addition, I would like to thank my committee members, Dr. Andreas Nebenführ, Dr. John Biggerstaff, and Dr. Elena Shpak, for their valuable advice about my research and their insights during the preparation and finalization of my thesis. I would also like to recognize the current and past members of the von Arnim lab, including Bijoyita Roy, Byung-Hoon Kim, Justin Vaughn, Xiuhua Han, Fujun Zhou, and Jongchan Woo, whose generosity and friendship made working in the lab an enjoyable and memorable experience. iii ABSTRACT Bioluminescence is a phenomenon in which chemical energy is converted into light energy. Here, the oxidation of a luciferin substrate, catalyzed by a luciferase enzyme, results in the emission of a photon. This biological process is exploited in a technology referred to as Bioluminescence Resonance Energy Transfer (BRET). As its name implies, BRET depends on a nonradiative energy transfer event that occurs between a donor luciferase and an acceptor fluorophore. Fusion of the donor and acceptor molecules to a protein(s) of-interest allows one to identify and monitor molecular events, such as protein interactions or hormone binding events, based solely on the spectral properties of the light produced. A primary goal of this research was to use BRET to investigate protein-protein interactions. Traditionally, BRET has been used to detect intermolecular interactions between protein pairs. To this end, BRET was applied to explore putative interactions between transcriptional regulators essential for organ polarity and floral development in Arabidopsis, FILAMENTOUS FLOWER (FIL), YABBY5 (YAB5), and LEUNIG (LUG). Results indicated that FIL and LUG are likely to interact in planta, supporting previous hypotheses that they function together within a protein complex. BRET has also been utilized to identify intramolecular, conformational changes that occur following a ligand-protein binding event. Hormone-binding sensors, in particular, have seen considerable success and are now used to monitor changes in small molecule concentrations within a cellular context. To identify whether BRET-based sensors are applicable to plant hormone studies, three sensors were created, incorporating AUXIN BINDING PROTEIN 1 (ABP1), GIBBERELLIC ACID INSENSITIVE iv DWARF 1A (GID1A), and CHICKEN THYROID HORMONE RECEPTOR, LIGAND BINDING DOMAIN (cTRα-1 LBD). Data obtained indicated that with modifications, single-molecule BRET sensors may be suitable for use in plants. Finally, luminescence imaging allows one to observe BRET as it occurs over a period of time. An imaging system was set up and used to monitor BRET occurring within transgenic Arabidopsis seedlings harboring either hRLUC-YFP or hRLUC constructs. Taken together, results from each of these studies highlight the versatility and sensitivity of BRET, and show that it can be used to monitor molecular events in a variety of applications. v TABLE OF CONTENTS Chapter I.........................................................................................................................1 BRET: An overview of the technology and its application to biological research.............1 Introduction.................................................................................................................1 Detection of protein-protein interactions..................................................................2 Bioluminescence resonance energy transfer (BRET) technology .............................3 Applications of BRET.................................................................................................7 BRET as a tool for measuring intramolecular conformational changes.....................7 Imaging BRET signals........................................................................................... 11 Questions addressed in this work............................................................................... 12 Chapter II...................................................................................................................... 14 Applying BRET to measure interactions between transcriptional corepressor proteins found in Arabidopsis thaliana ....................................................................................... 14 Introduction............................................................................................................... 14 YABBYs are important regulators of abaxialization and floral organ identity........ 15 LEUNIG shares similar functions with YABBY proteins ...................................... 17 Additional evidence for a FIL-LUG interaction ..................................................... 18 Methods .................................................................................................................... 19 In vivo BRET experiments .................................................................................... 19 Results ...................................................................................................................... 21 Discussion................................................................................................................. 23 Chapter III .................................................................................................................... 25 Developing novel BRET-based hormone sensors to detect conformational changes in proteins ......................................................................................................................... 25 Introduction............................................................................................................... 25 Selection of hormone-binding proteins ...................................................................... 26 Auxin binding protein 1 (ABP1)............................................................................ 26 Gibberellic acid insensitive dwarf (GID1A)........................................................... 28 Chicken thyroid hormone receptor, ligand-binding domain (cTR LBD)................. 30 Methods .................................................................................................................... 32 Constructing hormone sensors..............................................................................