The photobiology of cyanobacteriochrome Tlr0924 A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy (PhD) in the Faculty of Life Sciences 2014 Anna Hauck 2 | P a g e Table of Contents Abbreviations ........................................................................................................................... 6 List of figures .......................................................................................................................... 10 List of tables ........................................................................................................................... 12 Abstract .................................................................................................................................. 13 Declaration ............................................................................................................................. 14 Copyright statement .............................................................................................................. 14 Acknowledgements ................................................................................................................ 15 Preface to the alternative format .......................................................................................... 16 Chapter 1 – Introduction ............................................................................................. 17 1. Introduction ....................................................................................................................... 18 1.1 Basis of light absorbance ............................................................................................ 19 1.2 Photoreceptor structure ............................................................................................. 25 1.3 Classes of photoreceptors .......................................................................................... 29 1.3.1 Flavinoid photoreceptors ................................................................................... 29 1.3.1.1 Cryptochromes ........................................................................................... 29 1.3.1.2 Blue light sensors using flavin adenine dinucleotide ................................. 31 1.3.1.3 Light, oxygen or voltage sensors ................................................................ 32 1.3.2 Isomerisation based photoreceptors ................................................................. 33 1.3.2.1 Retinylidene proteins ................................................................................. 33 1.3.2.2 Xanthopsins ................................................................................................ 36 1.3.2.3 Phytochromes ............................................................................................. 38 1.3.2.4 Cyanobacteriochromes............................................................................... 40 1.4 Application .................................................................................................................. 48 1.5 Thesis aims and objectives.......................................................................................... 50 Chapter 2 – Experimental Set-up ............................................................................. 51 2. Spectroscopy ...................................................................................................................... 52 2.1 Stationary absorption spectroscopy ........................................................................... 54 2.1.1 UV/Visible absorption spectroscopy .................................................................. 55 2.1.2 Low-temperature UV/Visible absorption spectroscopy ..................................... 55 2.1.3 Infrared absorption spectroscopy ...................................................................... 57 2.2 Time-resolved absorption spectroscopy..................................................................... 59 3 | P a g e 2.2.1 Ultrafast visible absorption spectroscopy ........................................................... 60 2.2.2 Ultrafast infrared absorption spectroscopy ........................................................ 62 2.2.3 Flash photolysis ................................................................................................... 63 2.3 Data analysis ................................................................................................................ 64 2.4 Conclusion…………………………………………………………………………………………………………….. 64 Chapter 3 – Heterologous expression and spectral characterisation of the cyanobacteriochrome Tlr0924 ................................................. 65 3.1 Abstract ....................................................................................................................... 66 3.2 Introduction................................................................................................................. 67 3.3 Materials and methods ............................................................................................... 69 3.3.1 Plasmid construction ........................................................................................... 69 3.3.2 Protein expression .............................................................................................. 70 3.3.3 Protein purification ............................................................................................. 70 3.3.4 Spectroscopy ....................................................................................................... 70 3.4 Results ......................................................................................................................... 72 3.4.1 Expression and purification of Tlr0924 full-length protein ................................. 72 3.4.2 Stationary photoconversion properties .............................................................. 74 3.4.3 Importance of a second thioether linkage .......................................................... 76 3.4.4 Tlr0924 spectral sensitivities ............................................................................... 78 3.4.5 Chromophore dark reactions .............................................................................. 84 3.4.6 Temperature effects on Tlr0924 ......................................................................... 86 3.4.7 Cryobuffer compatibility ..................................................................................... 88 3.5 Discussion .................................................................................................................... 92 Chapter 4 – The photoinitiated reaction pathway of full-length cyanobacteriochrome Tlr0924 ......................................................... 94 4.1 Abstract ....................................................................................................................... 95 4.2 Introduction................................................................................................................. 96 4.3 Experimental procedures ............................................................................................ 99 4.3.1 Protein expression and purification .................................................................... 99 4.3.2 Ultrafast transient absorption spectroscopy ...................................................... 99 4.3.3 Laser flash photolysis ........................................................................................ 100 4.3.4 LED flash photolysis........................................................................................... 101 4.3.5 Cryotrapping ..................................................................................................... 101 4.3.6 Global analysis ................................................................................................... 102 4 | P a g e 4.4 Results ....................................................................................................................... 103 4.4.1 Ultrafast transient UV/Visible dynamics .......................................................... 103 4.4.2 Ultrafast transient IR dynamics ........................................................................ 105 4.4.3 Slow, thermally-driven dynamics ..................................................................... 106 4.4.4 Cryotrapping of intermediate states ................................................................ 112 4.5 Discussion ................................................................................................................. 114 Chapter 5 – Comprehensive analysis of the green to blue photoconversion of full-length CBCR Tlr0924 ......................... 118 5.1 Abstract ..................................................................................................................... 119 5.2 Introduction .............................................................................................................. 120 5.3 Materials and methods ............................................................................................. 122 5.3.1 Protein expression and purification ................................................................. 122 5.3.2 Ultrafast transient absorption spectroscopy .................................................... 122 5.3.3 Laser flash photolysis ........................................................................................ 123 5.3.4 Cryotrapping ....................................................................................................