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[Thesis Title] RICE UNIVERSITY By Anna Guseva A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE Master of Science APPROVED, THESIS COMMITTEE Jonathon Silberg Jonathon Silberg (Jun 8, 2020 14:43 CDT) Joff Silberg George Bennett George Bennett (Jun 11, 2020 21:17 CDT) George Bennett Caroline Ajo-Franklin HOUSTON, TEXAS June 2020 ABSTRACT Flavodoxin protein electron carriers: bioinformatic analysis and interactions with sulfite reductases by Anna Guseva Flavodoxins (Flds) are oxidoreductases that distribute electrons to different metabolic pathways through interactions with an array of partner proteins. The aim of my thesis is to understand Fld evolution, establish whether Flds are encoded within the same genomes as Fd-dependent sulfite reductases (SIRs), and demonstrate that a cellular assay can monitor Fld electron transfer (ET) to SIRs. Using bioinformatics, I identify numerous microbes whose genomes encode both Fld and SIR genes. Additionally, I show that Flds can support ET to SIR using a synthetic pathway where protein-mediated ET is monitored using the growth of an Escherichia coli auxotroph that depends upon Fld transferring electrons from a Fd:NADP+ reductase to SIR. My results represent the first evidence that Flds support ET to assimilatory SIRs. Additionally, they show how a synthetic ET pathway in cells can be leveraged to rapidly compare the ET efficiencies of different Flds. ii Acknowledgments While my advisor, Dr. Joff Silberg, is known for saying that PhD is not a sprint but a marathon, my accelerated Master’s program sometimes felt like a marathon that you run as if it were a sprint. Balancing my research with classes and other activities in order to complete this thesis would not be possible without the support of many mentors, members of the lab, friends, and family. First, I would like to acknowledge my advisor, Dr. Silberg, who guided me through the design and implementation of my research project, and was always open to discussing my ideas, questions, and concerns. I would especially like to thank Dr. Silberg for constantly being more optimistic about my results than I was. In our meetings, he helped me learn a lot about extracting useful information from the data that did not fit my expectations and adjusting my plans based on new results. I am very grateful that he also supported me in pursuing other projects in addition to my research, such as my participation in iGEM (International Genetically Engineered Machine) competition. Another person without whom I would not have even started my research project is PhD student Jordan Bluford, who has been my graduate student mentor. His mentorship allowed me to learn many essential skills and develop independence as a researcher. He has been remarkably patient in helping me fix my mistakes, improve my writing and presentation skills, and develop confidence in my work. Jordan also gave me a piece of advice that became, without a doubt, the most useful strategy in my thesis work: “Sometimes, when we float in the oceans of despair, it’s ok to just take a break.” iii At all stages of my project, I received valuable advice and mentorship from other members of the lab. I would especially like to thank Dr. Ian Campbell and Dr. Josh Atkinson for teaching me many of the techniques central to my thesis, and being extremely patient in helping me troubleshoot the problems with my experiments. I also thank Ilenne Del Valle for her amazing advice on data visualization and science communication. Other lab members and alumni also provided great help and support throughout my project: Tasfia Azim, Emm Fulk, Annelise Goldman, Dongkuk Huh, Jing Jin, Dimithree Kahanda, Prashant Kalvapalle, Jinyoung Kim, Jayaker Kolli, Li Chieh Lu, Andrea Padron, Kiara Reyes, Swetha Sridhar, Emily Thomas, Albert Truong, Jennifer Vu, and Liz Windham. I also want to thank Shyam Bhakta and Dr. David Zong from the Bennett Lab at Rice for teaching me many useful lab techniques. Outside of the lab, I would like to thank my committee members Dr. George Bennett, Dr. Caroline Ajo-Franklin, and Dr. Matthew Bennett. I also want to acknowledge the BioSciences department administrators Dr. Susan Cates and Dr. Rachael Eaton, who helped me keep track of completing the degree requirements. In addition, I want to thank many teachers, who helped me develop skills needed to complete this thesis. Especially Dr. Beth Beason and Dr. Collin Thomas, for individually caring about each student and making their classes extremely interesting. Finally, I thank my family and friends for supporting me throughout my project. iv My last and special thanks goes to the friends who helped maintain the creative atmosphere of our research building, Keck Hall, by founding and contributing to the Keck Museum of Modern Arts, as well as Keck Hall Book club – some of the many initiatives that provided great comfort and inspiration. v Contents Acknowledgments ............................................................................................. iii Contents ............................................................................................................. vi List of Figures .................................................................................................. viii List of Tables ...................................................................................................... x Abbreviations ..................................................................................................... xi Introduction ......................................................................................................... 1 1.1. Flavodoxins are electron carriers with diverse metabolic roles ................... 1 1.2. Flavodoxins evolved to replace ferredoxins in iron limitation and oxidative stress conditions ................................................................................................ 5 1.3. In E. coli, flavodoxins have diverse roles in metabolism ............................. 9 1.4. Insights into flavodoxin structure and interactions have come from in vitro studies ............................................................................................................. 12 1.5. Fld is a promising component for biotechnology and bioelectronics ......... 14 1.6. Selections can be used to accelerate studies of protein electron carriers 15 1.7. Hypothesis tested in this research ............................................................ 18 Methods ............................................................................................................. 21 2.1. Materials ................................................................................................... 21 2.2. Plasmid construction ................................................................................ 22 2.3. Auxotroph growth assay ........................................................................... 22 2.4. Flow cytometry ......................................................................................... 23 2.5. Construction of the sequence similarity network and analysis of organism distribution ....................................................................................................... 24 2.6. Statistics ................................................................................................... 26 Results .............................................................................................................. 27 3.1. Bioinformatics: identifying flavodoxins that might interact with sulfite reductases ....................................................................................................... 27 3.2. Constructs for testing flavodoxin-mediated electron transfer to sulfite reductases ....................................................................................................... 39 3.3. Cyanobacterial flavodoxins support electron transfer to ferredoxin- dependent sulfite reductases .......................................................................... 41 vi vii 3.4. RFP fusion can be used to measure intracellular flavodoxin concentrations ........................................................................................................................ 45 Future directions .............................................................................................. 56 4.1. Investigating the interactions of flavodoxins with cyanobacterial sulfite reductases ....................................................................................................... 56 4.2. Designing protein switches using flavodoxins .......................................... 57 4.3 Investigating the relationship between cycling efficiency and electron transfer rates measured in vitro ....................................................................... 58 References ........................................................................................................ 60 Appendix A........................................................................................................ 70 Appendix B........................................................................................................ 74 Appendix C........................................................................................................ 75 Appendix D........................................................................................................ 76 vii List of Figures Figure 1. The mechanism of ET by FMN ........................................................... 3 Figure 2.
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