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PDF (Otey Finalthesis.Pdf) STRUCTURAL AND FUNCTIONAL EXPLORATION OF AN ARTIFICIAL FAMILY OF CYTOCHROMES P450 Thesis by Christopher R. Otey In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CALIFORNIA INSTITUTE OF TECHNOLOGY Pasadena, California 2007 (Defended October 30th, 2007) ii © 2007 Christopher R. Otey All Rights Reserved iii ACKNOWLEDGEMENTS The best feature of CalTech is the diversity and intelligence of the people one has an opportunity to work and interact with. The broad perspectives and vast knowledge base allows one to learn and grow in different ways each and every day. I have developed a lot of great friendships that I hope will last long into the future and I want to thank everyone I have come in contact with for how they have helped me grow as a person. In the laboratory, everyone’s assistance has been invaluable but I especially want to thank a few individuals. I want to start with Joff Silberg, who provided a great deal of mentorship during the early stages of my research and was invaluable in providing guidance and inspiration. I am thankful I could always go to him with questions, to get ideas and to get back on track. Kaori Hiraga was a joy to work with and developed SISDC and was extremely helpful in constructing the chimeric P450 library, both times we made it! I must thank the ‘theoretical’ folks in the lab; Jeff Endelman, Allan Drummond, Jesse Bloom, Chris Voigt and Chris Snow. Their assistance has proven to me once and for all that they do exist. I must especially thank Jeff Endelman and Chris Voigt for their direct contributions in the form of logistic regression tools and the SCHEMA algorithm. Michelle Meyer and I shared a lot of ideas and frustrations having very similar projects which was always a comfort. Matt Peters did a wonderful job of bringing a lot of us together in collaboration and provided invaluable chemistry knowledge and great support. I would like to thank all of the other P450 team members, Edgardo Farinas, Uli Schwaneberg, Pat Cirino, Jorge Rodriguez, Peter Meinhold, Matt Peters, and Mike Chen. The collaborative iv nature of this team throughout the years is something we should all be proud of. Everyone here provided help in the form of knowledge, ideas, materials and friendship. Without their assistance I would have been unable to accomplish and learn all that I have with cytochromes P450. I also had the pleasure to work with some undergraduate and rotation students. James Ross for his help during a summer in which he assisted in the development of many HPLC methodologies and reminded me just how smart a young college student can be. I want to also thankNatalie Kruk, Ward Walkup and Kiowa Bower for assistance with high-throughput characterization of the chimeric P450 library. A huge thanks goes to Geethani Bandara who fulfills so many roles in the lab. Not only does she fight the endless battle against lab entropy but also offers her support, friendship and an occasional helping hand in the lab. I was on the receiving end of each and every one of these and am very grateful for it. I also want to thank Marco Landwehr for his help at all levels. He is a great friend and colleague and was with me in the trenches on many of the experiments and analyses described here. He and his wife, Dani Dieterich, provided a ton of support outside the lab as well for which I will always be grateful. I want to thank Frances Arnold, for allowing me the opportunity work in her lab. Frances supplied much of the inspiration for this project and through this process I developed a great respect for her ability to drive a project forward. Observing her management style and focused analytical ability has allowed me to take much more away from my graduate school experience than I think one gets in the average lab. v I would also like to thank the Croal family. Graciela and Tim have made me feel completely welcome in their family and have provided constant support as if I were a son. Megan and Jimmy have been another set of siblings for me and always chimed in to make me laugh or remind me that I am very important. All of their love and support will never be forgotten. I would like to thank my brother, Clayton, for his inspiration and reminding me how great you can be at something when you truly love it. My Mother and Father have always provided constant love and support. They given me a solid foundation and provided a much-appreciated safety net which has allowed me to continue my education for so very long. They have served as role models with their own many accomplishments and are always there when I need them. Finally I want to thank my lovely girlfriend, Laura, who has offered constant support, patience and love. She continues to be my muse. vi ABSTRACT Protein families are comprised of numerous sequences that adopt a similar three- dimensional structure and functional properties. The superfamily of cytochromes P450 are an excellent example of a common structural scaffold being utilized for a variety of biological functions. This functional diversity is achieved in Nature through millions of years of evolution to create new and diverse sequences. We have used site-directed recombination guided by the computation algorithm SCHEMA to create an artificial family of cytochromes P450 in the laboratory. Members of this family possess unique properties such as altered activity profiles, increased thermostability and the ability to accept new substrates. We developed screening tools for the rapid analysis of hundreds of individual P450s. These high-throughput assays include the 4-aminoantipyrine (4-AAP) assay which is capable of detecting the hydroxylation of an aromatic ring. High-throughput carbon monoxide binding facilitates the rapid detection of P450s that correctly incorporate a heme cofactor and are thus properly folded and potentially functional. Finally, a substrate binding assay which measures a spectral shift that occurs when a substrate binds in a P450 active site is described. Fourteen double-crossover chimeras created from the bacterial P450s CYP102A1 and CYP102A2 were constructed to calibrate the P450 scaffold for SCHEMA, a computational algorithm used to minimize structural disruption in chimeric proteins. We found that only chimeras with high levels of structural disruption as measured by SCHEMA were vii unfolded. Among the fourteen chimeras we also observed three different activity profiles based on peroxygenase kinetic assays with the substrates p-nitrophenoxydodecanoic acid (12-pNCA), 2-phenoxyethanol and allyloxybenzene. We applied this calibration to create an artificial family comprising ~3,000 chimeric heme P450 proteins that correctly fold and incorporate a heme cofactor by recombining three cytochromes P450 at seven crossover locations chosen to minimize structural disruption. Members of this protein family differ from any known sequence at an average of 72 and by as many as 109 amino acids. Most (>73%) of the properly folded chimeric P450 heme proteins are catalytically active peroxygenases; some are more thermostable than the parent proteins. A multiple sequence alignment of 955 chimeras, including both folded and not, was analyzed using logistic regression analysis (LRA) to identify key structural contributions to cytochrome P450 heme incorporation and peroxygenase activity and suggests possible structural differences between parents CYP102A1 and CYP102A2. Thirty-four members of this artificial family were assayed for functional diversity on a set of eight substrates. P450 chimeras were able to exceed the parents in total activity on all eight substrates and were grouped into five different groups based on activity profiles using K-means clustering. Activity profiles on eight substrates were then performed in high throughput to produce a data set of 330 chimeras. The mean percent standard deviation of the activity assays showed the reproducibility of these high-throughput data and further analysis may reveal information about sequence-structure-function relationships. viii The products of the catalytic reactions of four chimeric P450s with substrates of human P450s, some of which are drug compounds, were analyzed by HPLC in order to determine their identity. Chimeras were able to produce authentic human metabolites of chlorzoxazone, zoxazolamine and propranolol, showed peroxidase acitivty on 4- aminobiphenyl and produced an unknown product with tolbutamide. Finally, the peroxygenase activity of a mutant P450 heme domain is able to be further altered and enhanced using directed evolution. After two rounds of directed evolution and screening with the 4-AAP assay, we found mutants with altered substrate specificities and an overall enhancement of activity. The design and high-throughput methodologies described here can be used to create artificial protein families and to discover new and useful protein sequences. Like natural protein families, artificial protein families can be used to identify regions of protein sequence and structure that are important for folding and function. This is especially useful for analyzing protein families with few members or for validating tools for structure prediction and for protein sequence-structure-function analysis. Artificial protein families are also rich in sequence diversity and can provide sources of novel protein function. Using the high-throughput methodologies described here, chimeric P450s with enhanced activity, altered activity profiles, and the ability to hydroxylate drug-like compounds to produce authentic human metabolites
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