Investigating Protein-Protein Interactions in the Chlorophyll Biosynthesis Pathway Sarah Louise Hollingshead A thesis submitted for the degree of Doctor of Philosophy Department of Molecular Biology and Biotechnology University of Sheffield December 2013 Summary Summary The production of chemical energy from light energy is arguably the most important reaction known, which makes photosynthesis one of the most important processes on Earth. Nearly all life depends on the energy derived from light, and it is this process that supports Earths oxygenated atmosphere. Photosynthesis is initiated by the absorption of light, which is performed by specialised pigment molecules known as the chlorophylls. These are highly specialised pigment molecules that belong to the tetrapyrrole family, which also includes vitamin B12, sirohaem and haem. Chlorophyll is synthesised via a series of chemical reactions collectively known as chlorophyll biosynthesis. Whilst the majority of enzymes required for chlorophyll biosynthesis are known and have, to some extent, been characterised, one enzyme, the magnesium protoporphyrin IX monomethylester cyclase (cyclase), remains an enigma. This enzyme catalyses the formation of the 5th isocyclic ring, altering the colour of the tetrapyrrole molecule from red to green. Despite being known for over 60 years, only one subunit of the cyclase has been identified. This is the catalytic or AcsF subunit, which contains a distinctive di-iron motif and is absolutely conserved across all known oxygenic photosynthetic organisms. One of the focuses of this work was to identify other subunits of the cyclase. This led to the discovery of Ycf54, a protein that is highly conserved among oxygenic photosynthetic organisms, which is essential for chlorophyll accumulation in Synechocystis sp. PCC6803. The latter half of this thesis focuses on investigating the structural and functional characteristics of Synechocystis Ycf54; a protein that had previously not been investigated. This work lead the identification of three residues in Ycf54 (D39, F40 and R82), which are required for protochlorophyllide synthesis and chlorophyll formation in Synechocystis, and the observation that these residues are all required for Ycf54 to interact with the catalytic (AcsF) subunit of the cyclase. Additionally, the crystal structure of wild type Synechocystis Ycf54 to a resolution of 1.2 Å, and the structures of two Ycf54 mutants (A9G and R82A), which have an in vivo phenotype were obtained using molecular replacement. Furthermore this work presents the first investigations into the previously unknown protein Slr0483 in Synechocystis. This protein contains the conserved C-terminal membrane anchoring CAAD (Cyanobacterial Aminoacyl-tRNA synthetases Appended Domain) domain and is found in all of the oxygenic photosynthetic organisms investigated. The experiments reported in this thesis show that Slr0483 is essential for photosystem stability and accumulation in Synechocystis and that this protein interacts with the chlorophyll biosynthesis enzymes protoporphyrin IX oxidase, protoporphyrin IX methyltransferase, the i Summary cyclase subunit Sll1214 and the geranylgeranyl reductase ChlP, as well as the haem biosynthesis enzyme ferrochelatase. Leading to the hypothesis that Slr0483 may serve as a membrane anchor, localising the enzymes required for chlorophyll and haem biosynthesis, to the sites where these tetrapyrroles are required in the thylakoid membrane (i.e. next to the sites of photosystem and cytochrome assembly). i Acknowledgements Acknowledgements Firstly, I would like to thank my supervisor Professor Neil Hunter FRS for his continued support and advice throughout the four years I’ve been with the lab. Thanks in particular for not rejecting my rather late plea for a PhD, for jetting me off to several exotic conference locations and for lots of useful scientific discussions. In relation to this work I’d like to thank Drs: Daniel Canniffe for introducing me to Synechocystis, lending me so many of his strains and teaching me how to use the HPLC, Phil Jackson for all his invaluable help, time and patience with the mass spectrometry, Roman Sobotka for sharing his knowledge on all things Synechocystis and Amanda Brindley for all her help and advice with protein purification. A huge thank you to Sophie Bliss for all her help with the crystallography and introducing me to crystal trials, polishing cover slips, CCP4 and PyMOL (such fun!), Dave Armstrong for doing the NMR analysis on my mystery pigment (I couldn’t have done that without you) and Jana Kopecna for her help with clear native gels. Thanks to the whole of E12/E11 in its entirety for being a lovely bunch of people to work with and for making work enjoyable. In particular, I would like to thank Mo and Lizzy for keeping everything going. Jack, Dan, Paul, Lizzie and Gooch for all their great lab banter and the Dave’s for generally being great chaps. Thanks for Katie for keeping me fuelled with lunch, Starbucks coffee and muffins. Finally, thanks to all of the friends I’ve met in Sheffield over the last three years. Especially to Sophie, Abi, Simon, Katie and many bottles of sauvignon blank, for keeping me sane with our Wednesday drinking sessions and for helping me escape from the algae to go procure ice-creams and duck watch in the park. ii Table of contents Table of contents Summary __________________________________________________________________________________________________ 1 Acknowledgements ______________________________________________________________________________________ 2 1. Introduction ____________________________________________________________________________________________ 1 1.1 Photosynthesis __________________________________________________________________________ 1 1.2 The origins of photosynthesis and photosynthetic bacteria _____________________ 3 1.2.1 The evolution of photosynthesis _______________________________________________ 6 1.3 Synechocystis as a model organism __________________________________________________ 9 1.4 Tetrapyrroles____________________________________________________________________________ 9 1.4.1 Formation of δ-aminolaevulinic acid ___________________________________________ 15 1.4.2 Condensation of δ-aminolaevulinic acid to porphobilinogen ________________________ 21 1.4.3 Porphobilinogen deaminase and uroporphyrinogen III synthase _____________________ 23 1.4.4 Uroporphyrinogen III to coproporphyrinogen III __________________________________ 27 1.4.5 Coproporphyrinogen III to protoporphyrinogen IX ________________________________ 28 1.4.6 Protoporphyrinogen IX to protoporphyrin IX_____________________________________ 31 1.4.7 The branch-point of haem and chlorophyll biosynthesis ___________________________ 33 1.4.8 S-adenosyl-L-methionine Mg-protoporphyrin IX methyltransferase __________________ 46 1.4.9 Mg-protoporphyrin IX monomethylester cyclase _________________________________ 49 1.4.10 Protochlorophyllide reductase _______________________________________________ 55 1.4.11 Divinyl reductase __________________________________________________________ 62 1.4.12 Geranylgeranyl reductase and chlorophyll synthase ______________________________ 64 1.5 Aims ______________________________________________________________________________________67 2. Materials and methods _____________________________________________________________________________ 69 2.1 Standard buffers, reagents and media _____________________________________________69 2.2 E. coli strains and plasmids ___________________________________________________________69 2.2.1 Chemically competent E. coli cells _____________________________________________ 69 2.2.2 Transformation of chemically competent E. coli cells ______________________________ 70 2.3 Synechocystis strains _________________________________________________________________70 2.3.1 Transformation of Synechocystis sp PCC6803 ____________________________________ 70 2.3.2 Construction of Synechocystis knock-out mutants ________________________________ 71 2.4 Nucleic acid manipulation ____________________________________________________________71 2.4.1 Preparation of plasmid DNA __________________________________________________ 71 2.4.2 Polymerase chain reaction (PCR) ______________________________________________ 71 2.4.3 Restriction enzyme digests ___________________________________________________ 72 iv Table of contents 2.4.4 Agarose gel electrophoresis of DNA ____________________________________________ 72 2.4.5 Recovery of DNA from agarose gels ____________________________________________ 72 2.4.6 Ligation of DNA into vectors __________________________________________________ 73 2.4.7 DNA sequencing ____________________________________________________________ 73 2.4.8 Reverse transcription PCR ____________________________________________________ 73 2.5 Analysis of protein_____________________________________________________________________ 74 2.5.1 Polyacrylamide gel electrophoresis (PAGE) ______________________________________ 74 2.5.2 Western blot analysis of protein _______________________________________________ 74 2.5.3 Immunodetection __________________________________________________________ 75 2.5.4 Determination of protein concentration _________________________________________ 75 2.5.5 Analysis of proteins by mass spectrometry: whole solution digest ____________________ 76 2.5.6 Analysis of proteins by mass spectrometry: in-gel digestion _________________________ 77 2.5.7 Analysis of protein complexes