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Structure and Energetics of RNA - Protein Interactions for HIV RREIIB Targeting Zinc Finger Proteins

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Structure and Energetics of RNA - Protein Interactions for HIV RREIIB Targeting Zinc Finger Proteins Georgia State University ScholarWorks @ Georgia State University Chemistry Dissertations Department of Chemistry 7-1-2008 Structure and Energetics of RNA - Protein Interactions for HIV RREIIB Targeting Zinc Finger Proteins. Subrata H. Mishra Follow this and additional works at: https://scholarworks.gsu.edu/chemistry_diss Part of the Chemistry Commons Recommended Citation Mishra, Subrata H., "Structure and Energetics of RNA - Protein Interactions for HIV RREIIB Targeting Zinc Finger Proteins.." Dissertation, Georgia State University, 2008. https://scholarworks.gsu.edu/chemistry_diss/22 This Dissertation is brought to you for free and open access by the Department of Chemistry at ScholarWorks @ Georgia State University. It has been accepted for inclusion in Chemistry Dissertations by an authorized administrator of ScholarWorks @ Georgia State University. For more information, please contact [email protected]. STRUCTURE AND ENERGETICS OF RNA - PROTEIN INTERACTIONS FOR HIV RREIIB TARGETING ZINC FINGER PROTEINS by SUBRATA H. MISHRA Under the Direction of Dr. Markus W. Germann ABSTRACT RNA - protein interactions constitute a vital part of numerous biochemical processes. In the HIV life cycle, the interaction of the viral protein Rev and the Rev Responsive Element (RRE), a part of unspliced HIV RNA, is crucial for the propagation of infectious virions. Intervention of this interaction disrupts the viral life cycle. Rev - RRE interaction initially occurs at a high affinity binding site localized to a relatively small stem loop structure called RREIIB. This binding event has been well characterized by a variety of biochemical, enzymatic and structural studies. Our collaborators have previously demonstrated the efficacy of zinc finger proteins, generated by phage display, in the specific targeting of RREIIB. We have shown that the binding of these zinc finger proteins is restricted to the bulge in stem loop IIB that Rev also targets. Currently these proteins bind RREIIB with dissociation constants in the nanomolar range. We have employed a wide assortment of biophysical techniques such as gel shift assays, circular dichroism, isothermal titration calorimetry and NMR structural studies to further investigate this interaction. Several mutants of the zinc finger proteins and the RNA were also studied to delineate the parts of the protein secondary structure as well as the role of specific side chains in this interaction. We have generated a solution structure of the RREIIBTR RNA bound zinc finger protein, ZNF29G29R, which displayed the highest affinity to this RNA. This has allowed us to shed further light on the molecular basis of this RNA - protein interaction and provides input for further refinement in our structure guided phage display. INDEX WORDS: HIV, Rev Responsive Element, RNA, protein structure, isothermal titration calorimetry, RNA protein interactions, zinc finger protein, NMR structure, molecular recognition. STRUCTURE AND ENERGETICS OF RNA - PROTEIN INTERACTIONS FOR HIV RREIIB TARGETING ZINC FINGER PROTEINS by SUBRATA H. MISHRA A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the College of Arts and Sciences Georgia State University 2008 Copyright by Subrata H. Mishra 2008 STRUCTURE AND ENERGETICS OF RNA - PROTEIN INTERACTIONS FOR HIV RREIIB TARGETING ZINC FINGER PROTEINS by SUBRATA H. MISHRA Chair: Dr. Markus W. Germann Committee: Dr. W. David Wilson Dr. Kathryn B. Grant Electronic Version Approved by: Office of Graduate Studies College of Arts and Sciences Georgia State University August 2008 iv Dedicated to my parents and Mark. v Table of Contents Dedication iv List of Tables ix List of Figures x List of Abbreviations xiv CHAPTER I Overvie AIDS and HIV 1 HIV replication cycle 1 Nuclear export of unspliced HIV mRNA 4 RRE – Rev interaction 6 Strategies to inhibit RRE – Rev interaction 8 Design of RREIIB binding zinc finger proteins 9 Dissertation objectives 10 References 11 CHAPTER 2 Production and purification of zinc finger proteins and RNA Introduction 16 Protein production, purification and yields 16 15 Protein folding 20 19 Protein stability 24 23 Salt concentration 24 23 vi Protein storage 25 24 RREIIBTR and mutants 26 25 Conclusion 27 26 References 28 CHAPTER 3 Preliminary analysis of RREIIBTR - ZNF interactions Introduction 29 Structural changes in RREIIBTR on ZNF binding: CD 30 Imino NMR 31 30 Free RREIIBTR 32 31 RREIIBTR – ZNF complex 35 34 RREIIBTR – ZNF mutants 48 47 Protein tertiary structure dependent RNA binding 51 50 Temperature stability of the complex 52 51 Salt effects on the complex 53 52 Binding orientation 53 52 Tertiary structural changes in ZNF on binding RREIIBTR 58 57 Conclusion 60 59 References 62 61 CHAPTER 4 Energetics of RREIIBTR - ZNF binding Introduction 64 63 Binding constants from gel shift assays 64 63 vii Energetics from isothermal titration calorimetry 68 67 a. RREIIBTR – ZNF mutants 68 67 b. Specific heat of binding 73 71 c. Effect of salt, pH and osmolytes on RNA binding 79 78 d. RNA mutants – ZNF29G29R 85 83 Conclusion 87 86 References 89 88 CHAPTER 5 NMR structure of RREIIBTR bound ZNF29G29R Introduction 92 90 Overview of free ZNF29G29R structure 92 90 Resonance assignments for RREIIBTR bound ZNF29G29R 93 91 Backbone chemical shift differences between free and bound ZNF29G29R 101 99 Coupling constants for the torsion angle !: HNHA 104 102 Distance restraints: NOESY 106 104 Structure calculation and analysis 107 105 Final constraints used in structure determination 112 110 Conclusion 117 115 References 121 119 Summation 124 121 References 125 122 viii Appendices A - Site directed mutagenesis primers 126 123 B - PROPKA amino acid sidechain predictions 128 125 C - ITC thermodynamics data 129 126 ix List of Tables Chapter 2 Table 2.1 Zinc finger proteins and mutants. 18 Table 2.2 RREIIBTR variant sequences and modifications. 27 Chapter 4 Table 4.1 ITC binding data for RREIIBTR – ZNF mutants binding. 70 Table 4.2 Accessible surface area of protein, RNA and complex. 75 Table 4.3 Proton uptake/release at pH 7.0. 83 Table 4.4 Effect of osmolyte (sucrose) on ZNF29G29R – RREIIBTR binding. 84 Table 4.5 ITC binding data for RREIIBTR mutants and variants to ZNF29G29R. 86 Chapter 5 Table 5.1 List of NMR experiments used for the assignment and structure determination of the RREIIBTR bound ZNF29G29R. 99 Table 5.2 Chemical shift assignment table from SPARKY. 100 Table 5.3 Coupling constants from the HNHA experiment. 105 Table 5.4 Overview of the final 30 conformers. 113 x List of Figures Chapter 1 Figure 1.1 The HIV replication cycle. 2 2 Figure 1.2 Involvement of the host machinery in the nuclear export of unspliced and singly spliced mRNA to the cytoplasm. 6 Figure 1.3 Rev Responsive Element RNA. 7 7 Chapter 2 Figure 2.1 Zinc finger folding. 20 19 Figure 2.2 ZNF29 and mutants: protein folding. 23 22 Figure 2.3 Protein and NaCl elution profile of D-Salt ExcelluloseTM column. 25 24 Chapter 3 Figure 3.1 CD spectra of free RREIIBTR RNA (red) and ZNF29G29R complexed RNA (blue). 31 30 Figure 3.2 Imino NMR of free RREIIBTR. 32 31 Figure 3.3 Upper stem of RREIIBTR. 33 32 Figure 3.4 Assignment of RREIIBTR from its truncated versions. 34 Figure 3.5 Titration of RREIIBTR with ZNF29G29R followed by NMR at 298 K. 36 Figure 3.6 Comparison of RREIIBTR imino complex spectra with Rev and ZNF29G29R. 37 36 Figure 3.7 Identification of new imino region resonances. 38 37 xi Figure 3.8 RREIIBTR_G50_2AP. 40 39 Figure 3.9 RREIIBTR_G70_2AP. 41 40 Figure 3.10 RREIIBTR_G48_71_2AP. 42 41 Figure 3.11 RREIIBTR_G47_2AP. 43 42 Figure 3.12 RREIIBTR_U72_3MU. 44 43 Figure 3.13 RREIIBTR_C49U. 45 44 Figure 3.14 RREIIBTR_C69U. 46 45 Figure 3.15 RREIIBTR_32. 47 46 Figure 3.16 RREIIBTR – ZNF mutants imino spectra. 50 49 Figure 3.17 Structure dependent binding. 51 50 Figure 3.18 Thermal stability of the complex. 52 51 Figure 3.19 Effect of salt on the complex. 53 52 Figure 3.20 Plausible ZNF binding orientations. 54 53 Figure 3.21 Titration of unfolded ZNF28 with Co2+. 55 54 Figure 3.22 UV-visible (300-800 nm) spectrum of “cobalt loaded” ZNF28. 56 55 Figure 3.23 RREIIBTR – ZNF28 (100% cobalt folded). 57 56 Figure 3.24 RREIIBTR – ZNF28 (15% cobalt folded) titration. 57 56 Figure 3.25 RREIIBTR – ZNF29G29R binding model 58 57 Figure 3.26 Protein amide backbone. 59 58 Figure 3.27 15N HSQC overlay of free and RREIIBTR bound ZNF29G29R. 60 59 Figure 3.28 ZNF29G29R residues affected by RNA binding. 61 60 Figure 3.29 RNA phosphate backbone. 61 60 xii Chapter 4 Figure 4.1 ZNF29 – RREIIBTR gel shift assay. 67 66 Figure 4.2 Binding isotherm for ZNF29G29R titrated into RREIIBTR. 69 Figure 4.3 ZNF binding RREIIBTR in the major groove at the bulge. 71 Figure 4.4 Hydrogen bonding of the amino acid side chains in ZNF29. 72 Figure 4.5 Enthalpy - entropy compensation and specific heat of binding. 73 Figure 4.6 Enthalpy and entropy of all ZNF mutants binding RREIIBTR. 76 Figure 4.7 Electrostatic surface potential of ZNF29G29R and molecular volumes. 77 Figure 4.8 Effect of salt on ZNF – RREIIBTR binding. 80 78 Figure 4.9 Effect of pH on the free energy of binding. 82 80 Figure 4.10 Graphical representation of enthalpy, entropy and free energy of binding for all RNA mutants and variants with ZNF29G29R 87 ZNF29G29R. 85 Figure 4.11 The role of solvent in the enthalpy - entropy compensation phenomenon. 89 87 Chapter 5 Figure 5.1 NMR solution structure of free ZNF29G29R (pdb id: 2AB7). 93 91 Figure 5.2 HNCA magnetization transfer. 94 92 Figure 5.3 HNCA strip plot for the connectivity F7 - F14. 95 93 Figure 5.4 HBHA(CO)NH magnetization transfer. 96 94 Figure 5.5 HCCH TOCSY magnetization transfer.
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