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Production and Characterization of Arae I Production and Characterization of ArAE Family Members including Putative Efflux Transporters (PETs) from Bacteria and Aluminium Activated Malate Transporters (ALMTs) from Plants Antony James Palmer Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds School of Biomedical Sciences August 2016 II I The candidate confirms that the work submitted is his/her own and that appropriate credit has been given where reference has been made to the work of others. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. © 2016 The University of Leeds and Antony James Palmer The right of Antony James Palmer to be identified as Author of this work has been asserted by him in accordance with the Copyright, Designs, and Patents Act 1988 II Acknowledgements This thesis is dedicated to the memory of Steve Baldwin. Steve was the reason I came to Leeds and my inspiration to complete this thesis. I am sure that if he had still been able to participate in the preparation of this thesis, it would be much improved for his help. I hope he would have found it worthy. I will always remember his kindness and support, and if I can have even half of his relentless enthusiasm and positivity in life I will be doing well. I wish to thank those whose words and support meant so much, and those who have shared stories and memories of Steve and all those who attended the conference organised in Leeds. It was heartening to hear that this is not the end for his legacy. In particular I would like to thank Sheena Radford for encouraging words and Peter Henderson for being a calming influence as well as a repository of knowledge. And now to the more conventional acknowledgements. I wish to thank my supervisors for their guidance, clarity of thought, and constant patient support. Their constructive criticism, comments, and feedback, provided me with direction when I found myself lacking. The BBSRC should be thanked for their funding, organising events and trips, and facilitating PIPS. Thanks go to YCR for allowing me to come for an internship placement, and for Katherine Scott for accepting me. I learnt a lot. Members of Level 6 and Level 9 are thanked for creating an encouraging working environment Vincent Agboh, Dave Carrier, Peter Oatley, Catherine Qi, Amelia Lesiuk, Clair Phillips, Cheng Ma, Zhenyu Hao, Andrea Rawlings, Emily III Caseley, Tim Munsey, Ceasar Stanislaus, Lin Rong, Nita Shah, Maren Thompson, Craig Wilkinson, Jack Wright, Sarah Sabir. Continue eating cakes, playing squash, and playing games, all. You joined in with all our pranks and enhanced my competitive spirit, and remember that there’s nothing wrong with complicated. Special thanks go to David Sharples for superlative technical support, training on the cell disruptor, and performing the fermentations within this thesis. Even more special thanks go to Vincent Postis without whose knowledge and support I could not have learned and performed even half of the experiments herein. I could not have made it through without my friends. Vince A, Grace H, Jess H, Jess M, Jess B, Sarah S, Aaron M, Claudia M, Nita S, Dave C, Aimee L, Jamie K, Hannah S, Tom N, Jack G, James R, Ruth N, Laura M, Eleanor E, Fran R, Liz I, Chloe B, Rachel O, Debs R, Liz K. You’ve all made it special. In addition, much of my sanity has remained due to members of the Leeds ultimate frisbee team, and the wider frisbee community I’ve been able to be part of. As with all theses, many people provided important materials. Thanks go to Prof. George Lomonossoff (John Innes Centre) for providing the pEAQ-HT and pEAQ-GFP vectors for plant expression, Dr Carine De Marcos Lousa for providing Nicotiana benthamina seeds, Vince Agboh for preparing the pL51- ALMT plasmid, and Mike Harrison for fulfilling the thankless role of examiner of my transfer and providing useful comments and questions. A number of people here at Leeds should be thanked for their contributions to the experimental work presented here. James Ault performed protein identification by Mass Spectrometry, Nasir Khan assisted in CD measurements, IV Iain Manfield for use of the Optim in the Centre for Biomolecular Interactions, Chi Trinh supported the Crystallography, Martin Fuller provided technical assistance in EM, while firstly Robert Kolodziejczyk and more recently Maren Thompsen helped greatly with the SEC-MALLS operation and analysis and particularly Maren was a well of knowledge and advice. Samantha Aspinall, Martha Smith, and Lucy Parker in the Graduate School were invaluable on occasions too numerous to recall here, and also provided regular opportunities for free food and drink. I would like to thank my family, who have always had and unwavering belief in me and offered unconditional support. Vince, we have one last toast to make. V Abstract The focus of this study is proteins from the ArAE family, specifically two subfamilies: firstly, the ALMT family of plant membrane channels, which have numerous vital roles in plants, and secondly, the bacterial family first described as PET inner membrane exporters. Constructs were created for expression of firstly, the C-terminal domain (CTD) of wheat ALMT1 in E. coli and secondly, three full-length ALMTs (ALMT from wheat and ALMT5, ALMT9 from Arabidopsis) in Nicotiana benthamiana for structural and biochemical studies. Unfortunately, it appears that the CTD is not an independent soluble domain as originally thought, and this domain is now predicted to contain transmembrane helices, making it unsuitable for study in the manner planned. Similarly, production of full length ALMTs was unsuccessful as when extraction and purification was attempted, the protein degraded. Thirdly, a bacterial member of the ArAE family was expressed and characterised: AaeB from E. coli, along with its putative binding partner, AaeA. This was shown to form a complex with and be vital for the stability of AaeB. A strategy was devised for expression, solubilisation, and purification of these proteins and once they were obtained in pure form they were subjected to a range of biochemical and structural experiments. Microcrystals of AaeA were produced, towards a strategy for structural determination by X-ray crystallography. The first Electron Microscopy examination was performed on complexes of AaeB, and negative stain classes were produced. The first in silico homology model of AaeA has been produced and validated by CD spectroscopy, providing a range of insights. The complex formed by AaeA and AaeB has also been probed by crosslinking and SEC- MALLS analysis, suggesting a 6:2 or 6:3 stoichiometry. Together this has furthered our understanding of this poorly characterised membrane protein family and provided a set of clones and protocols for future studies. VI Table of Contents ACKNOWLEDGEMENTS ...................................................................................................... II ABSTRACT .......................................................................................................................... V TABLE OF CONTENTS ......................................................................................................... VI LIST OF FIGURES .............................................................................................................. XIII LIST OF TABLES ................................................................................................................. XX ABBREVIATIONS ............................................................................................................. XXII CHAPTER 1 INTRODUCTION .............................................................................................. 1 Compartmentalisation and Membranes ........................................................................................2 1.1.1 Biological membranes and membrane proteins ...................................................................2 1.1.2 Transport across membranes ...............................................................................................4 1.1.3 Channels and transporters ....................................................................................................5 1.1.3.1 Channels .....................................................................................................................5 1.1.3.2 Transporters ...............................................................................................................8 ArAE Family of Acid Exporters (including plant ALMTs) ...............................................................12 ALMT Family Members in Plants, Function, Topology, Mechanism, and Structure .....................14 1.3.1 Food security.......................................................................................................................15 1.3.2 Aluminium Resistance .........................................................................................................16 1.3.2.1 Overcoming aluminium toxicity on acid soils ...........................................................16 1.3.2.2 TaALMT1 in Aluminium Resistance ..........................................................................16 1.3.2.3 Other ALMTs involved in aluminium-resistance ......................................................17 1.3.3 Structural and topological studies of TaALMT1 and AtALMT1 ...........................................18 VII 1.3.3.1 C-terminal domain ..................................................................................................
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