The Cycle of Protein Engineering: Bioinformatics Design of Two Dimeric Proteins and Computational Design of a Small Globular Domain DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Venuka Durani, M.Sc. Graduate Program in Chemistry The Ohio State University 2012 Dissertation Committee: Thomas J. Magliery, Advisor Ross E. Dalbey Karin Musier-Forsyth William C. Ray Copyright by Venuka Durani 2012 Abstract The protein folding problem is an ongoing challenge, and even though there have been significant advances in our understanding of proteins, accurately predicting the effect of amino acid mutations on the structure and stability of a protein remains a challenge. This makes the task of engineering proteins to suit our purposes labor intensive as significant trial and error is involved. In this thesis, we have explored possibilities of better understanding and if possible improving some bioinformatics and computational methods to study proteins and also to engineer them. A significant portion of this thesis is based on bioinformatics approaches involving consensus and correlation analyses of multiple sequence alignments. We have illustrated how consensus and correlation metrics can be calculated and analyzed to explore various aspects of protein structure. Proteins triosephosphate isomerase and Cu, Zn superoxide dismutase were studied using these approaches and we found that a significant amount of information about a protein fold is encoded at the consensus level; however, the effect of amino acid correlations, while subtle, is significant nonetheless and some of the failures of consensus approach can be attributed to broken amino acid correlations. We also successfully engineered thermostabilized and active variants of the triosephosphate isomerase protein using correlation, phylogeny, and consensus information. ii In another project, the DNA binding domain of the E. coli Cra protein was used to evaluate the accuracy of some computational predictions. Based on the experimental data we came to the conclusion that computational prediction of the exact effect of a mutation on the structure and especially on the stability of a protein is challenging, and even the best computational tools available today have substantial room for improvement. We are carrying out extensive mutagenesis of the DNA binding domain of the E. coli Cra protein to compile a database of biophysical data to aid further development of computational techniques. The studies described above involved making several mutants of proteins and we also discuss the development of a new plasmid vector system that was used to clone many of these mutants. iii Dedication ॥ नम ्परमम ् ॥ -knowledge is the supreme goal- To Mumma and Papa; the two exemplary chemists, devoted teachers, and loving parents who inspired this journey. iv Acknowledgments Accomplishing the work reported in this thesis and also the actual writing of this thesis has come with a lot of stress and a lot of heartache but also with a lot of hope and a lot of joy. There are many people who have helped me through this journey and who were here to share with me the ups and downs of the rollercoaster that is grad-school, but talking about each and every one of them is beyond the scope of this acknowledgement! I would, however, like to specifically thank some of the individuals who have left a lasting impression on my life. First and foremost I would like to thank my advisor Dr. Tom Magliery for giving me the opportunity to work in his lab. When I joined the lab it was at a breaking point in terms of space and I was officially the third graduate student to join that year. I am truly happy that Tom found a place for me in his group. I thank him for being a great teacher, for having great expectations from me, and for never letting me give up. I would also like to thank Dr. James Hopper for helping me trouble-shoot some of the problems while working with S. cerevisiae. Even though he was my mentor for only one afternoon, I learnt a lot from him and his help came at a very crucial time in my graduate career. Brandon was working on the TIM project since before I joined the group and I thank him for sharing it with me. Vivek and Shila have also been great collaborators on the projects that I have shared with them. I thank Ted for his phenomenal contribution to v the FruR project and Kim for the sincerity in her work. I also thank Dr. Will Ray and his former graduate student Dr. Hatice Gulcin Ozer for several stimulating discussions on bioinformatics methods. The past several years that I have spent in Magliery Lab have taught me invaluable lessons in Chemistry and in life. For that I thank each and every “Maglieryite” that I have had the pleasure of sharing this lab with. I thank Christina for showing me how to use a cuvette washer and for some very amusing conversations when I first joined the group. I thank Jason and Mohosin for showing me two polar opposite ways of dealing with grad-school and with life. I thank David for being a constant source of entertainment by just being himself! I thank Danielle and Sarah for all the delicious baked goods in group meetings and Vidya for being her organized and dignified self. I thank Chau for playing the “who gets to lab first” game with me and Lihua for introducing me to her various absurd “truths of life”. My fellow 3040 inhabitants deserve a special mention for helping me get through many a day and for putting up with me and my mess. I am especially grateful to Brinda for practically being my sister in the lab, for supporting me through many rough days and for being there to share with me the simple pleasures of successful experiments. I thank Brandon for all the gossip and entertainment and also for keeping us on our toes in anticipation of his next practical joke. I thank Nick for never ceasing to amuse me and Kim for crazy stories from her life. I also thank Ted, my one and only undergrad, for reintroducing me to optimism. I would not have been here without the support of my family, especially my parents. They gave me my life. They gave me my dreams and the strength to go chasing vi after them. They also gave me my sister who has always been there for me. Without the support of my parents and my sister, I would have never made it this far! I also thank my mother in-law and father in-law for welcoming me into their family and for flying all the way across the globe to visit me and to witness a glimpse of my grad-school life. I left my family behind and travelled to a different continent in pursuit of education. However, I have found an extended family on this end of the planet and I am thankful for their support over the years. Especially Nammo, Kammo, Shubham, Yashas, Vivek, Brinda and others who made Columbus a home away from home. I have to especially acknowledge the little kids in Columbus - Sahana, Sohan and Aashu - for making me their “Vanku Aunty”. I also thank the Durani and Goyal family members in USA for welcoming me into their homes. Last but not the least; I thank my husband, Akshat, for waiting for me patiently all these years and for believing in me more than even I do. He is my life and I could not have accomplished this work without his undying support and unconditional love. vii Vita 2001................................................................Kendriya Vidyalaya, IIT Powai, Mumbai 2006................................................................Integrated MSc in Chemistry, IIT Bombay 2006 to present ..............................................Graduate Associate, Department of Chemistry, The Ohio State University Publications Sullivan BJ, Nguyen T, Durani V, Mathur D, Rojas S, Thomas M, Syu T, Magliery TJ, “Stabilizing Proteins from Sequence Statistics: The Interplay of Conservation and Correlation in Triosephosphate Isomerase Stability” Journal of Molecular Biology, (in press) Sullivan BJ, Durani V, Magliery TJ, “Triosephosphate isomerase by consensus design: dramatic differences in physical properties and activity of related variants” Journal of Molecular Biology, 2011, 413, 195-208. Prajapati RS, Sirajuddin M, Durani V, Sreeramulu S, Varadarajan R, “Contribution of cation-pi interactions to protein stability” Biochemistry, 2006, 45, 15000-15010. Chakraborty K, Durani V, Miranda ER, Citron M, Liang X, Schleif W, Joyce JG, Varadarajan R, “Design of immunogens that present the crown of the HIV-1 V3 loop in a conformation competent to generate 447-52D-like antibodies” Biochemical Journal, 2006, 399, 483-491. Fields of Study Major Field: Chemistry Specialization: Biological Chemistry viii Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iv Acknowledgments............................................................................................................... v Vita ................................................................................................................................... viii Table of Contents ............................................................................................................... ix List of Tables ................................................................................................................... xvi List of Figures ................................................................................................................