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Biochemistry in Bacterioferritin

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Biochemistry in Bacterioferritin BIOCHEMISTRY IN BACTERIOFERRITIN by Uthaiwan Suttisansanee A thesis presented to the University of Waterloo in fulfillment of the thesis requirement for the degree of Master of Science in Chemistry Waterloo, Ontario, Canada, 2006 © Uthaiwan Suttisansanee 2006 I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners. I understand that my thesis may be made electronically available to the public. ii ABSTRACT Bacterioferritin, an iron storage protein having a 24-subunit (α24) quaternary structure, was used as a model for the study of host-guest interactions and guest encapsulation, making use of its spherical cage-like structure. A hexahistidine-affinity tag fused to the C-terminus of each bacterioferritin subunit was constructed. The C-terminus of each subunit points toward the inside of the cavity, while the N-terminus is exposed on the surface of the protein. The hexaHistag was able to form strong interactions with a nickel-nitrilotriacetic acid linked dye molecule (guest) and this interaction was used in attempts to develop a principle to control guest molecule encapsulation within the spherical cavity of the 24-mer bacterioferritin protein molecule. The procedure involved (1) subunit dissociation under acidic pH, (2) affinity controlled dye-Histag binding with exposed C-terminal (His)6 residues and (3) reassociation of the subunits at neutral pH. The encapsulation conditions involving step 1 and 3 were studied preliminarily using laser light scattering to measure size (hydrodynamic radius) of the protein particle with apoferritin as a model system as it resembles the size and structure of bacterioferritin. In order to encapsulate guest molecules, the emptied shell of bacterioferritin was generated by site-directed mutagenesis resulting in ferroxidase- as well as heme-free bacterioferritin mutants (E18A/M52L/E94A), and these mutants were used to examine protein stability before conducting encapsulation experiments. However, wild-type bacterioferritin possessed highest stability in maintaining its multisubunit structure; hence, it was used for the encapsulation studies. It was found that 100% bacterioferritin with His6-tag at the C-terminus, and a combination of 60% bacterioferritin with His6-tag at the C-terminus and 40% bacterioferritin without His6-tag at the C-terminus yielded similar amounts of encapsulated iii guest molecules. This suggested that all hexahistidine at the C-terminus were not equally available for dye molecule binding. iv ACKNOWLEDGEMENTS I would like to extend my sincere thank to Dr. John Honek, my supervisor, for giving me the opportunity to work on this project. His advice, guidance, patience, and time are very appreciated. I also would like to thank Dr. Elisabeth Daub for her advice on protein engineering, Dr. Zhending Su for his advice on mass spectroscopy, and Liz Gargatsougias for information on bacterioferritin. I would like to give special thanks to the Meiering Lab for providing access to their Nanosizer ZS instrument, to Peter Stathopulos for sharing his knowledge of laser light scattering, to the Palmer Lab for providing a PTI Fluorometer, and to Shenhui Lang for her advice on fluorescence measurement. I also wish to acknowledge the Thai Government, Thailand for financial support during my graduate studies. I am grateful to Nicole Sukdeo, Pei Hang, and Paula Walasek for their expertise and their patience in teaching me how the lab works. I also would like to thank Christine Hand, Danish Khan, and Kadia Mullings for their support and encouragement. I cannot say anything else except “you guys are the best”. I would like to thank my family, Sacorn, Jewhiang, Rossukon, and Sasirada for their understanding and support. I am lucky that I was born in this family. I also would like to thank Platong for the courage and caring. In addition, I would like to thank my housemate, Ming Bao, for her cooking during a past year. v TABLE OF CONTENTS Page ABSTRACT……………………………………………………………………………….. iii ACKNOWLEDGEMENTS……………………………………………………………… v TABLE OF CONTENTS………………………………………………………………… vi LIST OF TABLES………………………………………………………………………... x LIST OF FIGURES……………………………………………………………………..... xiii LIST OF ABBREVIATIONS……………………………………………………………. xxv CHAPTER 1: INTRODUCTION 1.1: Bacterial Iron Homeostasis…………………………………………………………. 1 1.1.1: Role of Iron in Living Organisms…………………………………………… 1 1.1.2: Iron Transporters……………………………………………………………..3 1.1.3: Iron Storage Proteins and Their Applications………………………………..8 1.1.4: Regulatory Role of Iron..……………………………………………………. 23 1.2: Escherichia coli Bacterioferritin…………………………………………………….. 25 1.2.1: Discovery of Bacterioferritin………………………………………………... 25 1.2.2: Overall Structure…………………………………………………………….. 26 1.2.3: Heme Binding Site…………………………………………………………... 31 1.2.4: Iron Core Formation………………………………………………………… 34 1.3: Nanotechnology and Encapsulation………………………………………………… 40 1.3.1: The Host…………………………………………………………………….. 40 1.3.2: The Guest…………………………………………………………….…….... 49 1.3.3: Linkage……………………………………………………………………… 51 1.3.4: Applications of Encapsulation Processes…………………………………… 56 1.4: Possible Aspects for Bacterioferritin Encapsulation………………………………. 57 1.5: Summary of Experimental Objectives Achieved…………………………………... 64 vi CHAPTER 2: CONDITIONAL TESTS FOR DISSOCIATION AND REASSOCIATION OF APOFERRITIN SUBUNITS USING LIGHT SCATTERING TECHNIQUES 2.1: Literature Research on Dissociation and Reassociation of Horse Spleen Apoferritin………………………………………………………………………………… 66 2.2: Reagents, Materials, and Equipment ……………………………………………… 72 2.3: Experimental Protocols……………………………………………………………… 73 2.4: Results and Discussion ……………………………………………………………… 76 2.4.1: Effect of Protein Concentration…………………………………………….. 76 2.4.2: Effect of pH…………………………………………………………………. 78 2.4.3: Effect of Electrolytes………………………………………………………... 79 2.4.4: Effect of Glycerol…………………………………………………………… 80 2.4.5: Effect of Incubation Time on Dissociation…………………………………. 81 2.4.6: Effect of Reassembly Time…………………………………………………. 82 2.5: Conclusions…………………………………………………………………………... 83 CHAPTER 3: OVEREXPRESSION AND PURIFICATION OF FERROXIDASE CENTER- AND HEME-FREE BACTERIOFERRITIN 3.1: Literature Research on the Ferroxidase Center- and Heme-free Bacterioferritin …………………………………………………………………………… 85 3.2: Reagents, Materials, and Equipment ………………………………………………. 87 3.3: Experimental Protocols……………………………………………………………… 90 3.3.1: Protocols for Control Experiments………………………………………….. 90 3.3.2: Protein Engineering…………………………………………………………. 93 3.3.3: Protein Expression, Induction, and Purification..…………………………… 97 3.4: Results and Discussion ……………………………………………………………… 101 3.4.1: Control Experiments on Protein Stability Conditions………………………. 101 3.4.2: Cloning of Wild-type and Bacterioferritin Mutants………………………… 107 3.4.3: Overexpression and Purification of Wild-type and Bacterioferritin Mutants.. 108 3.4.4: Protein Stability of Wild-type and Bacterioferritin Mutants…………………113 3.5: Conclusions……………………………………………………………………………120 vii CHAPTER 4: OVEREXPRESSION, PURIFICATION, AND ENCAPSULATION PROPERTIES OF C-TERMINAL HIS6-TAGGED BACTERIOFERRITIN 4.1: Literature Research on C-terminal Extension and Encapsulation of Bacterioferritin……………………………………………………………………………. 121 4.2: Reagents, Materials, and Equipment……………………………………………….. 125 4.3: Experimental Protocols……………………………………………………………… 125 4.3.1: Protein Engineering…………………………………………………………. 125 4.3.2: Protein Expression, Induction, and Purification ……………………………. 127 4.3.3: Identification of Dye Molecule……………………………………………… 128 4.3.4: Preparation for Encapsulation Experiments ………………………………... 134 4.3.5: Determination of the Fluorescence Quenching………………………………141 4.3.6: Measurement of Heme Groups Incorporated in Bacterioferritin …………… 142 4.3.7: Determination of the Presence of Iron Atoms in Bacterioferritin ………….. 143 4.3.8: Determination of the Number of Dye Molecules Encapsulated into Bacterioferritin………………………………………………………………. 143 4.4: Results and Discussion ……………………………………………………………… 144 4.4.1: DNA Cloning and Protein Purification……………………………………… 144 4.4.2: Dye Identification…………………………………………………………… 146 4.4.3: Optimum Conditions for Encapsulation of Bacterioferritin………………… 152 4.4.4: Control Experiments for Encapsulation by Bacterioferritin………………… 168 4.4.5: Fluorescence Quenching of the Dye Encapsulated Bacterioferritin………… 177 4.4.6: Heme-incorporated Encapsulated Bacterioferritin……….............................. 178 4.4.7: Iron Storage in Bacterioferritin ……………………………………………... 179 4.4.8: Dye Molecules Encapsulated in Bacterioferritin …………………………… 179 4.5: Conclusions…………………………………………………………………………... 180 CHAPTER 5: SUMMARY AND FUTURE WORK 5.1: Summary………………………………………………………………………………183 5.2: Future Work…………………………………………………………………………. 185 REFERENCES……………………………………………………………………………. 187 Appendix 1: Flow Chart for QuikChange® Site-directed Mutagenesis PCR……….. 203 Appendix 2: Flow Chart for Site-directed Mutagenesis of bFT-E94A ……………… 204 Appendix 3: Flow Chart for Protein Purification…………………………………….. 205 viii Appendix 4: Sequencing Results for Wild-type and Variants of Bacterioferritin……206 Appendix 5: Mass Spectrometric Results……………………………………………….217 Appendix 6: Spectrophotometric Results for Wild-type and Variants of Bacterioferritin……………………………………………………………. 227 Appendix 7: Gel Filtration Chromatographic Results (Superose 6) of Wild-type and Variants of Bacterioferritin…………………………………………. 232 Appendix 8: Calculation of Extinction Coefficient for Dye Molecules in Pro-Q® Sapphire 365 Oligohistidine
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