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Redesign of Calcium-Regulated Protein Aequorin Towards the Development

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Redesign of Calcium-Regulated Protein Aequorin Towards the Development Redesign of calcium-regulated protein aequorin towards the development of a novel ion bioreporter A Thesis submitted to University College London for the degree of Doctor of Philosophy by Evlampia-Kyriaki Dimitriadou Department of Biochemical Engineering University College London UCL April 2014 I, Evlampia-Kyriaki Dimitriadou confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. - 2 - I dedicate this work to my loving parents, Sophia and Panagiotis - 3 - Abstract This thesis aimed to design novel sensor proteins that can identify and measure various metal ions in vivo and in situ . Metal ions play key role in the metabolism of the cell, and monitoring of calcium has helped interrogate cellular processes such as fertilisation, contraction and apoptosis. Real-time monitoring of more divalent metal ions like zinc and copper is required to gain much needed insight into brain function and associated disorders, such as Alzheimer’s and Parkinson’s disease. Aequorin is a calcium-regulated photoprotein originally isolated from the jellyfish Aequorea victoria . Due to its high sensitivity to calcium and its non-invasive nature, aequorin has been used as a real-time indicator of calcium ions in biological systems for more than forty years. The protein complex consists of the polypeptide chain apoaequorin and a tightly bound chromophore (coelenterazine). Trace amounts of calcium ions trigger conformational changes in the protein, which in turn facilitate the intermolecular oxidation of coelenterazine and concomitant production of CO 2 and a flash of blue light. Aequorin’s light emitting reaction can also be triggered by a range of other divalent and trivalent cations, leading however to significantly lower light yields. Based on aequorin’s promiscuity towards other ions, this project tested the hypothesis that aequorin’s preference for certain cations could be manipulated through mutations engineered in one or more of the three calcium-binding loops (EF-hands). In order to test the hypothesis, the following six stages were performed: cloning of the apoaequorin gene for expression in E. coli ; development of a high-throughput assay for expression and measurement of bioluminescent activity in microwells; design of a library containing forty eight mutant variants of aequorin; screening of the library against seven metal ions; protein purification of wild-type aequorin and one selected mutant; analysis of activity and kinetics of purified wild type and one chosen mutant against all seven ions. This work produced mutants with shifted selectivity towards new metal ions at the cost of luminescence yield. The impact of mutations is analysed and it is suggested that one of the EF-hands (EF-I) is likely to serve as a gatekeeper to aequorin’s selectivity. It was also shown that at least one mutant utilised zinc ions (that wild type failed to utilise) to achieve low levels of bioluminescent activity. - 4 - Acknowledgements I would like to thank my supervisor, Professor Paul Dalby for his valuable input throughout the duration of my project. This work would not have been possible without his continued support. I would also like to thank my advisor, Professor Gary Lye for his advice and encouragement. Special thanks go to Dr Rigini Papi, Lazaros Batsilas and Dr Jean Aucamp for providing their insights and feedback of my work. I am also grateful to Dr Martina Micheletti for training on the robotic platform I used for all the screening work described in this thesis. I would also like to thank the PhD students of Foster Court and UB18, who made the labs feel like a second home. Finally, I am most grateful to and for my parents, Sophia and Panagiotis and my dear friends Issy, Maria, Stephanos, Rigini, Lazaros, Spyros, Erato, Jean, Zehra, Rosie and Maria W. for genuine love and support. Thank you for being patient with all my absenses and for waiting on the other side. - 5 - Contents Abstract..............................................................................................................4 Acknowledgements ...........................................................................................5 Contents .............................................................................................................6 List of tables ....................................................................................................15 List of figures...................................................................................................17 Abbreviations ..................................................................................................21 1 Introduction............................................................................................24 1.1 Bioluminescence..............................................................................24 1.1.1 Definitions....................................................................................24 1.1.2 Bioluminescence in nature...........................................................26 1.1.3 Aequorea victoria , the bioluminescent jellyfish ..........................27 1.1.4 Flash- and glow-type luminescence.............................................28 1.2 Bioreporters......................................................................................29 1.2.1 Definitions....................................................................................29 1.2.2 Need for ion bioreporters .............................................................30 1.2.3 Desirable characteristics for bioreporters.....................................31 1.2.4 Signal transduction methods ........................................................32 1.2.5 Current methods for intracellular ion monitoring ........................34 1.3 Aequorin: a calcium-sensitive photoprotein ...................................36 1.3.1 Introduction..................................................................................36 1.3.2 Uses of aequorin...........................................................................40 1.3.3 Properties of aequorin ..................................................................41 1.3.4 EF-hand motif: general features..................................................44 1.3.4.1 EF-hands and cooperativity......................................................46 1.3.5 Proposed mechanism for the bioluminescence reaction ..............47 1.3.6 Regeneration of aequorin.............................................................47 1.3.7 Ion selectivity of aequorin............................................................49 1.3.8 Other photoproteins......................................................................50 1.4 Coordination of metals in proteins...................................................51 1.5 Protein engineering ..........................................................................56 1.5.1 Rational design.............................................................................56 - 6 - 1.5.2 Directed evolution........................................................................57 1.5.3 Integration of random and rational approaches............................58 1.5.4 Metalloprotein engineering..........................................................59 1.6 Overview..........................................................................................60 1.6.1 Aequorin mutants as potential bioreporters .................................60 1.6.2 Potential activator ions for aequorin mutants...............................61 1.7 Aims and organisation of thesis.......................................................62 2 Materials and Methods..........................................................................64 2.1 Preparation of buffers and media.....................................................64 2.1.1 LB medium ..................................................................................64 2.1.2 LB agar.........................................................................................64 2.1.3 YT agar.........................................................................................65 2.1.4 SOC medium................................................................................65 2.1.5 Kanamycin ...................................................................................65 2.1.6 Ampicillin ....................................................................................65 2.1.7 X-gal.............................................................................................66 2.1.8 Tris-HCl buffer.............................................................................66 2.1.9 EDTA...........................................................................................66 2.2 Cell culture.......................................................................................67 2.2.1 E. coli strains................................................................................67 2.2.1.1 TOP10 electrocompetent ..........................................................67 2.2.1.2 BL21Star™(DE3).....................................................................67 2.2.2 Streaked plates .............................................................................67 2.2.3 Overnight cultures........................................................................68 2.2.4 Shake flask cultures......................................................................68 2.2.5 Glycerol stocks.............................................................................68
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