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Biophysical Characterization of Protein Interactions by ITC and NMR Xiaochen Lin University of Connecticut - Storrs, [email protected]

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Biophysical Characterization of Protein Interactions by ITC and NMR Xiaochen Lin University of Connecticut - Storrs, Xiaochen.2.Lin@Gmail.Com University of Connecticut OpenCommons@UConn Doctoral Dissertations University of Connecticut Graduate School 4-29-2016 Biophysical Characterization of Protein Interactions by ITC and NMR Xiaochen Lin University of Connecticut - Storrs, [email protected] Follow this and additional works at: https://opencommons.uconn.edu/dissertations Recommended Citation Lin, Xiaochen, "Biophysical Characterization of Protein Interactions by ITC and NMR" (2016). Doctoral Dissertations. 1058. https://opencommons.uconn.edu/dissertations/1058 Biophysical Characterization of Protein Interactions by ITC and NMR Xiaochen Lin, PhD University of Connecticut, 2016 Proteins are crucial in virtually all cell activities. The vast majority of proteins interact with other molecules to function properly. The investigation of biomolecular interactions calls for structural and thermodynamic information acquired by a variety of experimental techniques and analytical methods. This dissertation is mainly concentrated on two frequently used and powerful tools, Isothermal Titration Calorimetry (ITC) and Nuclear Magnetic Resonance (NMR) spectroscopy, to investigate protein interactions. p52 Shc, a well-studied cellular adaptor protein, is a positive regulator in the mitogen-activated protein kinases pathway. We have confirmed that Shc interacts with both, integrin tyrosine(s)-phosphorylated cytoplasmic tails and phosphotidylinositol lipids, through the same Phospho-Tyrosine Binding (PTB) domain. The binding to phosphorylated peptides is enthalpy driven and tighter, while Shc interactions with PtdIns are entropy driven and much weaker. Shc interactions with PtdIns and β3-derived peptides are only weakly competitive with partially overlapping bindings sites. We also observed thermodynamic indications for potential intramolecular interactions within Shc, which could be perturbed by the binding to the phosphorylated receptor. BTN3A1, an immunoglobulin superfamily member, was found recently to be critical in Vγ9Vδ2 T cell activation. We described a phosphoantigen prodrug approach with great potency in Vγ9Vδ2 T cell stimulation. Our NMR and ITC results revealed the intracellular binding of BTN3A1 to phosphoantigens. In addition, we have provided biophysical evidence indicating the involvement of membrane proximal region in phosphoantigen binding and potential structural changes within BTN3A1 intracellular region. By focusing on p52 Shc and BTN3A1, I hope to demonstrate how a combination of structural and thermodynamic knowledge can be utilized to answer many important questions in signaling transduction close to the cell membrane. Biophysical Characterization of Protein Interactions by ITC and NMR Xiaochen Lin B.S., China Pharmaceutical University, 2009 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Connecticut 2016 i Copyright by Xiaochen Lin 2016 ii APPROVAL PAGE Doctor of Philosophy Dissertation Biophysical Characterization of Protein Interactions by ITC and NMR Presented by Xiaochen Lin, B.S. Major Advisor ______________________________________________________________________ Olga Vinogradova Associate Advisor ___________________________________________________________________ Debra Kendall Associate Advisor ___________________________________________________________________ Andrei Alexandrescu Associate Advisor ___________________________________________________________________ Andrew Wiemer University of Connecticut 2016 iii Table of Contents Table of Contents .................................................................................................................. iv Acknowledgements ................................................................................................................ vi List of Figures ...................................................................................................................... viii List of Tables ......................................................................................................................... x List of Abbreviations .............................................................................................................. xi Chapter 1 ITC in Protein Interaction Study ............................................................................. 1 Principles of ITC ..................................................................................................... 1 Data Analysis of ITC ............................................................................................... 3 Practical Considerations in an ITC Experiment ....................................................... 7 Chapter 2 NMR spectroscopy in Protein Interaction Study ................................................... 10 Overview of NMR Spectroscopy ........................................................................... 10 Principles of NMR Spectroscopy........................................................................... 11 Chemical Shifts in Protein NMR ............................................................................ 13 1H-15N HSQC 2D Experiments .............................................................................. 14 Chapter 3 p52 Shc in Signaling............................................................................................ 15 Shc Family ............................................................................................................ 15 Shc in MAPK Pathway .......................................................................................... 16 Shc in PI3K/AKT Pathway .................................................................................... 17 Chapter 4 Phospho-Tyrosine(s) versus Phosphatidylinositol Binding in Shc Mediated Integrin Signaling .............................................................................................................................. 21 Introduction ........................................................................................................... 21 Shc Interaction with Phosphatidylinositols Tested Using Ins(1,4,5)P3 ....................... 24 Shc Interaction with Phosphatidylinositols Analysed by ITC Using PtdIns(4)P1 and PtdIns(4,5)P2 ...................................................................................................................... 27 PtdIns Binding Sites on Apo Shc PTB Surface Partially Overlap with Integrin Binding Sites ................................................................................................................................. 29 Story of Integrin Interactions with the Shc - PTB Domain versus Full Length Shc . 32 PtdIns Only Weakly Compete with β3-Peptides for Interaction with Shc ................ 34 Discussion ............................................................................................................ 38 Research Design and Methods ............................................................................. 42 Materials and Reagents .................................................................................... 42 Cloning and Protein Purification ........................................................................ 42 ITC Experiments ............................................................................................... 44 15N-HSQC Titration by NMR .............................................................................. 45 iv Chapter 5 Vγ9Vδ2 T cells in the Immune System ................................................................ 46 Overview of the Immune System in Humans ........................................................ 46 Lymphocytes: B Cells and T Cells ........................................................................ 47 Vγ9Vδ2 T Cells ..................................................................................................... 50 Activation of Vγ9Vδ2 T Cells by Phosphoantigens ................................................ 51 BTN3A1 in Activation of Vγ9Vδ2 T Cells............................................................... 55 Chapter 6 Do Phosphoantigens Bind to the Extracellular IgV Domain of BTN3A1? ............. 57 The Extracellular Binding Model of IgV-Phosphoantigen ...................................... 57 Crystal Structures of IgV Complexed with Phosphoantigens................................. 57 Mass Spectroscopy and SPR Analysis of the IgV-Phosphoantigen Interaction ..... 60 Chapter 7 Intracellular Binding of BTN3A1 to Phosphoantigens .......................................... 62 A POM-protected Phosphoantigen is Highly Potent in Vγ9Vδ2 Stimulation. ......... 62 The BTN3A1 PRY/SPRY (B30.2) Domain Directly Binds Phosphoantigens. ........ 65 The Intracellular Domain of BTN3A1 Undergoes Conformational Changes after Binding to Phosphoantigens. ............................................................................................ 75 Discussion ............................................................................................................ 78 Research Design and Methods ............................................................................. 79 Cloning and Purification of BTN3A1 Constructs ................................................ 79 ITC Experiments ............................................................................................... 80 15N-HSQC Titration by NMR .............................................................................. 80 Author Contributions ............................................................................................
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