“Utilisation of amber suppression/non- natural amino acid technology for protein engineering and cellular control” by Josephine Lydia Morris A Thesis submitted to Cardiff University for the degree of Doctor of Philosophy Cardiff University March 2013 i DECLARATION This work has not been submitted in substance for any other degree or award at this or any other university or place of learning, nor is being submitted concurrently in candidature for any degree or other award. Signed ………………………………………… (candidate) Date …………………… STATEMENT 1 This thesis is being submitted in partial fulfillment of the requirements for the degree of …………………………(insert MCh, MD, MPhil, PhD etc, as appropriate) Signed ………………………………………… (candidate) Date …………………… STATEMENT 2 This thesis is the result of my own independent work/investigation, except where otherwise stated. Other sources are acknowledged by explicit references. The views expressed are my own. Signed ………………………………………… (candidate) Date …………………… STATEMENT 3 I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loan, and for the title and summary to be made available to outside organisations. Signed ………………………………………… (candidate) Date …………………… ii Acknowledgements Firstly I’d like to thank my supervisors Dr Eric Tippmann and Professor Phil Stephens who have given me the opportunity to undertake the work presented here; provided crucial supervision throughout the research and preparation of this thesis. I would like to acknowledge Dr Sohie Shim, who performed the ESI-MS analysis on my protein samples; Dr Damien Murphy who performed the EPR spectroscopy and data analysis; Dr Elisabetta Canetta who performed the Raman spectroscopy; Dr E. Joel Loveridge for greatly assisting me during the circular dichroism measurements; Dr Pierre Rizkallah who assisted with protein crystal formation and collection and who performed x-ray diffraction at Diamond Light Source, Didcot. Without the support from the staff within the Schools of Chemistry and Dentistry the work presented would not have been possible and I appreciate their help. I am grateful to Professor Rudolf Allemann and Dr Dafydd Jones for the use of their laboratory equipment and helpful scientific comments. I’d like to thank Dr Alicja Antonczak and Dr Zuzana Simova for showing me the way when I first started and throughout the first and second year of my research. They were both a source of intellectual knowledge and friendship. I’d also like to thank the other members of Physical Organic Chemistry group, past and present, particularly Jamie Johnson and Azzedine Bouleghlimat, for their friendship and scientific discussions. Also, the Wound Biology Group within the School of Dentistry and members of Dr Dafydd Jones’ lab within the School of Bioscience, who have supported me and provided beneficial discussions. I would like to acknowledge the support and love provided by my Mum and Dad, my sisters, Alana and Naomi and my partner Gareth. In addition, I’d like to thank my friends, particularly, Dr Matt Edmundson, Dr Mike Gamble, Andy Hartley, Sam Reddington, Dr Amy Baldwin, and Seni Chanapai who have also provided support. iii Abstract The amber suppression technology is an intracellular methodology that allows position specific incorporation of a specific non-natural amino acid (NAA) into proteins using imported NAA-specific machinery during protein translation. The method has been utilised to incorporate over 50 NAAs into proteins (e.g. those that confer unique reactivity (and allow subsequent conjugation of additional factors), installation of post-translational modification mimics, modulation of protein function and those that aid in structural determination). Therefore, the aim of the work presented within this Thesis was to explore the use of this technology in novel applications; namely the installation and study of a reactive moiety within a defined environment and the creation of a ‘biological switch’ to control the production of the protein and subsequently a cellular phenotype. It was demonstrated that incorporation of the NAA, azidophenylalanine, within the hydrophobic pocket of T4 Lysozyme (T4LazF) could provide a protein scaffold to stabilise, shield and thereby allow exploration of the chemical reactivity of the photoreactive aryl azide moiety using various spectroscopic techniques. Specifically, electron paramagnetic spectroscopy of irradiated T4LazF demonstrated that the singlet phenyl nitrene species had been captured. To create a ‘biological switch’ to control the production of a protein and the subsequent cellular phenotype, the duality of the amber suppression method was the basis for the novel application rather than the chemical reactivity of the NAA. E. coli and mammalian cell motility readout systems were successfully created using flagellin (non-flagellate) and Rac1 (GTPase protein involved in lamellipodia production) knockout cell lines respectively in conjunction with specially created plasmid constructs. However, complexities regarding the implementation of amber suppression in order to control this motility via the presence/absence of NAA limited the functionality of these readout systems. The work presented has advanced the field of amber suppression and NAA technology by demonstrating that generating reactive intermediates derived from NAAs within a defined chemical environment of a protein provides a novel technique to generate and study highly reactive intermediates. In addition, it has been shown that the amber suppression technology has potential to act as a biological switch to control cellular responses. iv Table of contents 1 CHAPTER 1: INTRODUCTION ................................................................................ 1 1.1 Introduction ................................................................................................... 2 1.2 Translation ..................................................................................................... 3 1.3 Prokaryotic translation .................................................................................. 3 1.3.1 Ribosome ................................................................................................ 3 1.3.2 Initiation ................................................................................................. 4 1.3.3 Elongation .............................................................................................. 4 1.3.4 Termination ............................................................................................ 7 1.4 Eukaryotic translation ................................................................................... 7 1.4.1 Ribosome ................................................................................................ 7 1.4.2 Initiation ................................................................................................. 8 1.4.3 Elongation .............................................................................................. 8 1.4.4 Termination ............................................................................................ 8 1.5 Genetic code ................................................................................................ 10 1.5.1 Incorporation of pyrrolysine and selenocysteine ................................ 12 1.6 Aminoacylation ............................................................................................ 12 1.7 The aminoacyl tRNA synthetase .................................................................. 15 1.8 tRNA ............................................................................................................. 16 1.9 Identity elements ........................................................................................ 18 1.9.1 TyrRS-tRNATyr identity elements .......................................................... 19 1.10 Methods to incorporate NAAs .................................................................... 24 1.10.1 Chemical synthesis ............................................................................... 24 1.10.2 Semisynthesis ....................................................................................... 25 1.10.3 In vitro biosynthesis ............................................................................. 25 1.10.4 In vivo biosynthesis .............................................................................. 26 1.11 Expanding the genetic code ........................................................................ 27 1.11.1 Production of tRNA .............................................................. 30 1.11.2 Production of aaRSs specific to NAAs .................................................. 34 1.11.3 Applications of the amber suppression technology in E. coli .............. 37 1.11.3.1 Post-translational modification (PTM) mimics ............................. 37 1.11.3.2 Modulation of protein function .................................................... 38 1.11.3.3 Structural determination .............................................................. 39 1.11.3.4 Introduction of novel reactivity .................................................... 39 1.11.3.5 Probes ........................................................................................... 40 1.11.3.6 Other applications ........................................................................ 40 1.11.4 Amber suppression technology in mammalian cells ........................... 45 1.11.4.1 Production of tRNA ....................................................... 45 1.11.4.2 Production of aaRSs specific to NAAs ........................................... 46 v 1.11.4.3 Applications of the amber suppression technology in mammalian cells 48 1.12 Studying reactive intermediates using a defined protein environment ..... 50 1.12.1 p-azido-L-phenylalanine ....................................................................... 50 1.12.2 Aryl azide photochemistry ................................................................... 50 1.12.3 Spectroscopy of NAAs .......................................................................... 53 1.12.4 T4 Lysozyme ......................................................................................... 53 1.13 Controlling phenotype by amber suppression ............................................ 56 1.13.1 Bacterial motility .................................................................................. 56 1.13.1.1 Flagellar export ............................................................................. 58 1.13.2 Mammalian cell motility ...................................................................... 58 1.13.2.1 Amoeboid motion ......................................................................... 59 1.13.2.2 Metazoan motion ......................................................................... 59 1.13.2.3 Scratch wound model ................................................................... 61 1.13.2.4 Motility and the role of actin ........................................................ 63 1.13.2.5 GTPases and their role in cell migration ....................................... 65 1.13.2.6 Ras-related C3 botulinum toxin substrate 1 (Rac1) ...................... 65 1.13.2.6.1 Overall structure ....................................................................... 66 1.13.2.6.2 GTP exchange of Rac1 ............................................................... 68 1.13.2.6.3 Post-translational modification of C-terminal of Rac1 ............. 68 1.13.2.6.4 Signalling cascades elicited by Rac1 ......................................... 71 1.14 Aims ............................................................................................................. 74 2 CHAPTER 2: GENERAL MATERIALS AND METHODS ........................................... 75 2.1 Instruments ................................................................................................. 76 2.2 Materials ...................................................................................................... 76 2.2.1 Non-natural amino acids ...................................................................... 76 2.2.2 Bacterial antibiotics .............................................................................. 76 2.2.3 Buffers .................................................................................................. 77 2.2.3.1 Buffers for preparation of chemically competent cells ................... 77 2.2.3.2 Buffers for DNA preparation ............................................................ 77 2.2.3.3 Buffers for enzymatic reactions ....................................................... 78 2.2.3.4 Buffers for DNA electrophoresis ...................................................... 78 2.2.3.5 Buffers for microbiological media .................................................... 79 2.2.3.6 Buffers for sodium dodecyl sulphate polyacrylamide gel electrophoresis ................................................................................................ 79 2.2.4 Restriction enzymes ............................................................................. 81 2.2.5 Other enzymes ..................................................................................... 81 2.2.6 Escherichia Coli Cells ............................................................................ 81 2.2.7 Media ................................................................................................... 82 2.2.8 Plasmids ............................................................................................... 83 2.2.9 Primers ................................................................................................. 83 vi 2.3 Methods ...................................................................................................... 84 2.3.1 Microbiology ........................................................................................ 84 2.3.1.1 Preparation of electrocompetant E. coli cells .................................. 84 2.3.1.2 Preparation of chemically competent E. coli cells ........................... 84 2.3.1.3 Transformation of DNA in to electrocompetant E. coli .................... 85 2.3.1.4 Transformation of DNA in to chemically competent E. coli ............. 85 2.3.1.5 Preparation of bacterial stocks ........................................................ 85 2.3.1.6 Isolation of plasmid DNA .................................................................. 85 2.3.1.7 Motility assay .................................................................................... 87 2.3.2 Molecular Biology ................................................................................ 87 2.3.2.1 Polymerase Chain Reaction (PCR) .................................................... 87 2.3.2.1.1 Colony PCR .................................................................................. 87 2.3.2.1.2 General and Mutagenic PCR ....................................................... 88 2.3.2.2 Purification of DNA products ............................................................ 89 2.3.2.3 Phosphorylation of PCR products ..................................................... 90 2.3.2.4 Digestion of plasmid DNA by restriction enzymes ........................... 90 2.3.2.5 Dephosphorylation of DNA .............................................................. 90 2.3.2.6 Ligation ............................................................................................. 91 2.3.2.7 Agarose Gel electrophoresis ............................................................ 91 2.3.2.7.1 DNA electrophoresis standard .................................................... 91 2.3.2.8 DNA quantification ........................................................................... 92 2.3.2.9 DNA sequencing ............................................................................... 92 2.3.2.10 Non-natural amino acids .............................................................. 92 2.3.3 Protein Analysis .................................................................................... 92 2.3.3.1 Sodium dodecyl sulphate polyacrylamide gel electrophoresis ........ 92 2.3.3.1.1 SDS-PAGE electrophoresis standard ........................................... 94 2.3.3.1.2 Sample preparation for SDS-PAGE ............................................. 94 2.3.3.1.3 Visualisation of SDS-PAGE gels ................................................... 94 2.3.3.2 Concentration of protein .................................................................. 95 2.3.3.3 Determination of protein concentration ......................................... 95 3 CHAPTER 3: PHOTOCHEMISTRY OF ARYL AZIDES IN DEFINED PROTEIN ENVIRONMENTS ......................................................................................................... 96 3.1 Introduction ................................................................................................. 97 3.2 Materials and methods ............................................................................. 100 3.2.1 T4 lysozyme Expression...................................................................... 100 3.2.2 T4 lysozyme extraction ...................................................................... 100 3.2.3 T4 lysozyme purification .................................................................... 101 3.2.3.1 Nickel affinity chromatography ...................................................... 101 3.2.3.2 Size exclusion chromatography (SEC) ............................................ 101 3.2.4 T4 Lysozyme crystallisation ................................................................ 101 3.2.5 Spectroscopic analysis ........................................................................ 102 vii 3.2.5.1 Protein photolysis for EPR .............................................................. 102 3.2.5.2 Electroparamagnetic resonance spectroscopy (EPR) ..................... 102 3.2.5.3 Ultraviolet-visible spectroscopy ..................................................... 103 3.2.5.4 Fourier transform infrared spectroscopy ....................................... 103 3.2.5.5 Circular dichroism ........................................................................... 103 3.2.5.6 Raman spectroscopy ...................................................................... 103 3.3 Results ....................................................................................................... 104 3.3.1 Expression optimisation ..................................................................... 104 3.3.1.1 Plasmids pT4L153TAG/pAAT4L153TAG and pBKiodoRS ................ 104 3.3.1.2 Plasmids pETT4L153TAG and pSUPiodoRS ..................................... 108 3.3.2 Purification optimisation .................................................................... 115 3.3.2.1 Nickel affinity chromatography ...................................................... 115 3.3.2.2 Size exclusion chromatography ...................................................... 117 3.3.3 Investigation in to the expression of T4L153azF................................ 119 3.3.3.1 Using pSUPiodoRS to incorporate azF ............................................ 119 3.3.3.2 Using pAAazRS to incorporate azF ................................................. 119 3.3.3.3 Using pDULEazF to incorporate azF ............................................... 119 3.3.3.4 Comparison of plasmid systems, pET vs. pBAD .............................. 121 3.3.4 Expression and purification of WT T4L .............................................. 124 3.3.5 Fidelity of aaRS ................................................................................... 126 3.3.6 Expression and purification of T4L153azF ......................................... 126 3.3.7 Investigating the promiscuity of azRS ................................................ 129 3.3.8 Crystallisation of WT and mutant T4L ................................................ 129 3.3.9 Circular Dichroism of T4L ................................................................... 130 3.3.10 Spectroscopic evaluation of azido substituted T4L ........................... 132 3.3.10.1 EPR of T4L153azF ........................................................................ 132 3.3.10.2 Ultraviolet-visible spectroscopy ................................................. 134 3.3.10.2.1 UV-vis of amino acid, azF ........................................................ 134 3.3.10.2.2 UV-vis of T4L153azF ................................................................ 134 3.3.10.3 Infrared spectroscopy ................................................................. 139 3.3.10.4 Raman Spectroscopy .................................................................. 139 3.4 Discussion .................................................................................................. 142 3.4.1 T4L expression .................................................................................... 142 3.4.1.1 Plasmid systems ............................................................................. 142 3.4.1.2 Plasmid encoding orthogonal aaRS and tRNA ............... 145 3.4.1.3 Promiscuity of NAA-RSs .................................................................. 146 3.4.1.4 Fidelity of amber suppression ........................................................ 146 3.4.1.5 Functionality of mutant T4L ........................................................... 147 3.4.2 T4L purification .................................................................................. 148 3.4.3 Crystallisation of T4L .......................................................................... 149 3.4.4 EPR of T4L153azF ............................................................................... 150 viii 3.4.5 Further spectroscopic investigations ................................................. 156 3.4.5.1 CD spectroscopy ............................................................................. 156 3.4.5.2 Mass spectrometry ......................................................................... 156 3.4.5.3 UV vis spectroscopy ........................................................................ 157 3.4.5.4 IR spectroscopy .............................................................................. 159 3.4.5.5 Raman Spectroscopy ...................................................................... 160 3.5 Conclusion ................................................................................................. 161 4 CHAPTER 4: UTILISATION OF THE AMBER SUPPESSION TECHNOLOGY TO CONTROL A FUNCTIONAL BACTERIAL READOUT SYSTEM ....................................... 163 4.1 Introduction ............................................................................................... 164 4.2 Results ....................................................................................................... 167 4.2.1 Analysis of E. coli motility ................................................................... 167 4.2.1.1 Functional FliC gene is required for motility .................................. 167 4.2.2 Restoration of motility by plasmids-encoded FliC ............................. 170 4.2.2.1 Cloning FliC into pAA plasmid ......................................................... 170 4.2.2.2 Introduction of pAAFliC into non-motile strains ............................ 170 4.2.2.3 Cloning FliC into pQE60 plasmid .................................................... 174 4.2.2.4 Introduction of pQE60FliC into non-motile strains ........................ 174 4.2.3 Plasmid pFD313 and pFDFliC.............................................................. 177 4.2.3.1 Creation of the plasmid pFDFliC ..................................................... 177 4.2.3.2 Restoration of motility using pFDFliC ............................................. 178 4.2.4 Implementation of amber suppression in FliC ................................... 180 4.2.4.1 Introduction of TAG mutation into FliC .......................................... 180 4.2.4.1.1 Structural and positional considerations .................................. 180 4.2.4.1.2 Creation of plasmid pFDFliC239TAG ......................................... 182 4.2.4.2 Introduction of pFDFliC239TAG in to KAF95 strain ........................ 182 4.2.4.3 Introduction of pFDFliC239TAG in to Keio strain ........................... 182 4.2.4.4 Investigations into the control of motility via amber suppression technology ..................................................................................................... 185 4.3 Discussion .................................................................................................. 188 4.3.1 FliC gene is required for motility of E. coli ......................................... 188 4.3.2 Plasmids encoding FliC ....................................................................... 188 4.3.3 KAF95 could not be used to modulate motility via amber suppression 190 4.3.4 Keio had the potential to be used to modulate motility via amber suppression ...................................................................................................... 191 4.3.5 Implementation of amber suppression technology .......................... 192 4.3.5.1 Improving amber suppression ........................................................ 195 4.4 Conclusion ................................................................................................. 197 5 CHAPTER 5. DEVELOPMENT OF A FUNCTIONAL MAMMALIAN CELL READ OUT SYSTEM ..................................................................................................................... 198 5.1 Introduction ............................................................................................... 199 ix 5.2 Materials and methods ............................................................................. 201 5.2.1 Tissue culture plastic .......................................................................... 201 5.2.2 Instruments ........................................................................................ 201 5.2.3 Software ............................................................................................. 201 5.2.4 Molecular biology............................................................................... 201 5.2.5 Mammalian Cell Culture .................................................................... 202 5.2.5.1 Cell lines .......................................................................................... 202 5.2.5.2 Media .............................................................................................. 202 5.2.5.3 Recovery from cryostorage ............................................................ 204 5.2.5.4 Cell sub-culture ............................................................................... 204 5.2.5.5 Preparation of cells for cryostorage ............................................... 204 5.2.5.6 Cellular Transfection ....................................................................... 205 5.2.5.7 Creation of monolayer scratch wound ........................................... 205 5.2.5.8 Stable selection .............................................................................. 206 5.2.5.9 Creation of stable cell lines ............................................................ 206 5.2.5.9.1 Picking GFP-positive stably transfected colonies ..................... 206 5.2.5.10 Live Cell Imaging ......................................................................... 207 5.2.5.11 RNA extraction ............................................................................ 207 5.2.5.11.1 DNase treatment .................................................................... 208 5.2.5.11.2 Quantification of RNA ............................................................. 208 5.2.5.12 Reverse transcription polymerase chain reaction (RT-PCR) ....... 208 5.2.5.12.1 cDNA synthesis ........................................................................ 208 5.2.5.12.2 PCR amplification .................................................................... 209 5.2.6 Image Analysis .................................................................................... 211 5.2.6.1 Creation of merged image .............................................................. 211 5.2.6.2 Calculation of transfection efficiency ............................................. 211 5.2.6.3 Quantification of wound closure .................................................... 211 5.2.6.4 Quantification of the total distance moved and directionality of cells 212 5.2.7 Statistical analysis .............................................................................. 212 5.3 Results ....................................................................................................... 214 5.3.1 Analysis of control and Rac1 KO cell lines .......................................... 214 5.3.1.1 Loss of Rac1 alters cell morphology and proliferation ................... 214 5.3.1.2 Optimisation of scratch wound assay ............................................ 216 5.3.1.3 Normal cell migration is dependent on the expression of Rac1 .... 216 5.3.1.4 Quantitative analysis of wound closure ......................................... 219 5.3.1.5 Quantitative analysis of motility of individual cells ....................... 219 5.3.2 Restoration of motility to Rac1 KO cells ............................................ 222 5.3.2.1 Plasmid constructs for transfection in to Rac1 KO cells ................. 222 5.3.2.2 Creation of peGFP plasmid for transfection into Rac1 KO cells ..... 222 5.3.2.3 Introduction of plasmid-encoded Rac1 to Rac1 KO cells ............... 224 x