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University of London Thesis Z C REFERENCE ONLY UNIVERSITY OF LONDON THESIS Degree Year Name of Author Z C G f h j > COPYRIGHT This is a thesis accepted for a Higher Degree of the University of London. It is an unpublished typescript and the copyright is held by the author. All persons consulting the thesis must read and abide by the Copyright Declaration below. COPYRIGHT DECLARATION I recognise that the copyright of the above-described thesis rests with the author and that no quotation from it or information derived from it may be published without the prior written consent of the author. LOANS Theses may not be lent to individuals, but the Senate House Library may lend a copy to approved libraries within the United Kingdom, for consultation solely on the premises of those libraries. Application should be made to: Inter-Library Loans, Senate House Library, Senate House, Malet Street, London WC1E 7HU. REPRODUCTION University of London theses may not be reproduced without explicit written permission from the Senate House Library. Enquiries should be addressed to the Theses Section of the Library. Regulations concerning reproduction vary according to the date of acceptance of the thesis and are listed below as guidelines. A. Before 1962. Permission granted only upon the prior written consent of the author. (The Senate House Library will provide addresses where possible). B. 1962- 1974. In many cases the author has agreed to permit copying upon completion of a Copyright Declaration. C. 1975 - 1988. Most theses may be copied upon completion of a Copyright Declaration. D. 1989 onwards. Most theses may be copied. This thesis comes within category D. This copy has been deposited in the Library of l i d u This copy has been deposited in the Senate House Library, Senate House, Malet Street, London WC1E 7HU. Production of disulphide-bonded domains suitable for NMR structure determination: Application to the SRCR domains o f the lymphocyte receptor CD5 Alicia Acely Garza-Garcia University College London A thesis submitted for the degree of Doctor of Philosophy January 2005 1 UMI Number: U591717 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U591717 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract This work describes the development of a systematic methodology to overcome two of the difficulties commonly encountered when expressing eukaryotic domains in bacterial hosts, namely the failure to obtain folded protein in vivo and the low solubility of the expression product. This methodology made possible the production of samples of the first scavenger receptor cysteine rich (SRCR) domain of human CD5, /zCD5dl, with properties suitable for multidimensional NMR studies. The SRCR domains of hC D5 express in bacteria as insoluble aggregates. The aggregates were purified in order to perform the folding in vitro. Optimal conditions for folding were found using a novel systematic screen based on a fractional factorial design. In vitro folding yields were assessed using RP-HPLC and non-denaturing PAGE. The attainment of /zCD5dl protein samples of sufficiently high concentration to perform multidimensional NMR was achieved by performing rational apolar-to-polar mutations selected by analysis of a multiple sequence alignment. Eight single residue mutants were engineered and expressed. Four of them had better in vitro folding yields than the wild type and a double mutant was constructed by combination of the best behaved single mutations. This double mutant was used to determine the structure of the domain. NMR experiments at 298 K showed that some regions of /zCD5dl undergo conformational exchange on a microsecond to millisecond timescale, hampering the assignment of the resonance signals. Increasing the temperature to 318 K was found to greatly enhance the quality of the NMR spectra and enabled the assignment of more than 95% of the resonances. The solution structure of /zCD5dl was determined using standard interproton distance and dihedral angle-restrained molecular dynamics protocols. Forty percent of the residues were found to be in structurally well-defined regions, including all of the regular secondary structure features found for other members of SRCR superfamily. The remaining residues of the polypeptide appear to be distinctly less well ordered. 2 To those whose presence in my life makes me suspect that I am the most fortunate human in the Universe: Brigida, Gustavo and Mark 3 Acknowledgements I am indebted to many for the fact that this arduous process is about to reach its conclusion. Not just to those who directly contributed to the work presented here, but also to all those who supported me and gave me reasons to smile and to keep fighting to tame CD5 throughout these years. I am utterly grateful to the Mexican National Council for Science and Technology (CONACYT) for their financial support during my PhD. I wish to thank them and all the people of Mexico that made this possible. My thanks also to the Graduate School of UCL for providing me with a complementary scholarship for three years. It is a pleasure to thank my supervisor Prof Paul Driscoll for all his guidance, trust and support. I have learned from him more things than he will be willing to admit. A mole of thanks to my team mates, Dr Richard Harris and Dr Diego Esposito for assigning the NMR spectra of CD5dl and doing the structure calculations while I was still trying to tie some loose ends in the wet lab. A bit more thanks to Richard Harris in his position as NMR manager for setting up the NMR experiments and for sharing some of his knowledge on practical NMR with me. Many thanks to Prof Neil Barclay, Dr Marion Brown and Dr Namir Hassan at Sir William Dunn School of Pathology, Oxford University for providing us with the DNA of the extracellular region of CD5 and CD6. My gratitude to Prof Penny Handford, Dr Kristy Downing, Dr Jo O'Leary and Vroni Knott, at the Department of Biochemistry of Oxford University for advice on in vitro protein folding and inspiring conversation. Oodles of thanks to Dr Mark Williams for his critical review of my manuscript, his help and relentless discussion throughout my PhD. I am grateful to Dr Federico del Rio from whom I have learned a plethora of things about NMR spectroscopy, programming and human relationships. 4 I am very grateful to my lab mates for their friendship, help and support, for the things I have learned from them and for introducing me to the subtleties of English (and Scottish!) idiosyncrasy. Thanks to Andrew S, Beatriz, Brian, David, Diego, Gillian, Mike, Mark J, Matt, Richard, and Roger. Many thanks to Dr Snezana Djordjevic and my mentor Prof John Ladbury for their useful suggestions and interest in my project. I would like to thank my parents for their example, support and unconditional love. Thanks to my brother and to all my wonderful Mexican/Dominican/Swiss/Brazilian/ (and now) English family, for always being there for me, waiting for news on my progresses. On this side of the Pond I have met some wonderful people that have made all the difference in this voluntary exile of mine. Thanks to Abril, Beatriz M., Danielle, Gabriela, Gillian, Manuel, Richard and Virginia, for their enduring support and encouragement. A special thanks to Virginia and the Arechavala-Gomeza household for providing our larder with scrumptious Spanish food that filled not only our stomachs but also our spirits. Each and every day I am grateful for the friendship of a handful of individuals whose affection remains unaltered despite the enormous distance between us. Thanks to Aubin, Eva, Liliana, Ricardo, Rina and Yuki. Last but not least, thanks to my husband. He stoically put up with me and my neurosis since those days when I could not make the ligations to work. Thank you Mark, for being who you are and for remaining by my side in this process of becoming who I want to be. 5 Table of contents ABSTRACT___________________________________________________________________ 2 ACKNOWLEDGEMENTS_______________________________________________________ 4 TABLE OF CONTENTS_________________________________________________________ 6 LIST OF FIGURES_____________________________________________________________13 LIST OF TABLES_____________________________________________________________ 16 CHAPTER ONE CD5. AN ENIGMATIC MODULATOR OF LYMPHOCYTE ACTIVATION______________17 Abstract 17 Background 19 The B cell receptor for antigen 19 Structure of the T cell receptor for antigen and its mechanism of antigen recognition 21 Thymocyte maturation 23 Signal transduction by the antigen receptors 23 Lymphocyte surface glycoprotein CD5 24 CD5 protein structure 29 CD5 regulation in lymphocyte activation 29 CD5 stimulation can enhance the activation signals of the TCR in mature T cells 32 CD5 stimulation is able to inhibit the activation of T cells 32 CD5 increases the threshold for TCR stimulation during T cell maturation 33 CD5 inhibits the signalling events activated by BCR engagement and promotes the production of a cell survival cytokine 34
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