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Redbird RA D 2010.Pdf (4.946Mb) Identification of a protein kinase substrate in Sulfolobus solfataricus P2 Ruth Ann Redbird Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Biochemistry Peter J. Kennelly, Chair Richard Helm James Mahaney Renee Prater April 1, 2010 Blacksburg, VA Keywords: protein kinase, phosphorylation, Archaea, ATP Synthase Identification of a protein kinase substrate in Sulfolobus solfataricus P2 Ruth Ann Redbird Abstract Living organisms rely on many different mechanisms to adapt to changes within their environment. Protein phosphorylation and dephosphorylation events are one such way cells can communicate to generate a response to environmental changes. In the Kennelly laboratory we hope to gain insight on phosphorylation events in the domain Archaea through the study of the acidothermophilic organism Sulfolobus solfataricus. Such findings may provide answers into evolutionary relationships and facilitate an understanding of phosphate transfer via proteins in more elaborate systems where pathway disturbances can lead to disease processes. A λ-phage expression library was generated from S. solfataricus genomic DNA. The immobilized expression products were probed with a purified protein kinase, SsoPK4, and radiolabeled ATP to identify potential native substrates. A protein fragment of the ORF sso0563, the catalytic A-type ATPase subunit A (AtpA), was phosphorylated by SsoPK4. Full length and truncated forms of AtpA were overexpressed in E. coli. Additional subunits of the ATPase were also overexpressed and ATPase activity reconstituted in vitro. Phosphoamino acid analysis and MS identified the phosphorylation sites on AtpA. Several variants of AtpA were derived via site-directed mutagenesis and assayed for ATPase activity. Chemical cross-linking was employed to determine possible ATPase subunit interactions; tryptic digests of AtpA and its mutant variants were performed to examine protein folding. The phosphorylated-mimic variant of AtpA, T98D, resulted in an inactive ATPase complex as determined by ATPase activity assays and native-PAGE indicating potential phosphoregulation by SsoPK4 on enzyme activity. Ultimately, any findings would need verification with in vivo studies. iii Acknowledgements Let me start by thanking my advisor and mentor, Dr. Peter J. Kennelly, for giving me the chance to work in the lab first as a technician and then as a graduate student. “Dr. K” always found time for questions, input, and advice on all matters both professional and personal even after his life became hectic with department chair duties. Thanks for teaching me to “think like a scientist” and to become a better person all around. You have made a great impact on my life and it will not go unnoticed or underappreciated! Another round of thanks goes out to the people who have passed through the lab in my time at VT: graduate students Archana Mukhopadhyay, January Haile and Brian Jordan, undergraduates Andrew Chute and my advisees Laura Pompano and Joe Arthur. To Ben Potters and grad student Hanan Dahche, many thanks for “Salad Fingers”, Conan clips and random photo shoots with blue liquid – that must be science at work. You two made the last years a lot of fun. Special acknowledgement goes to Dustin Hite for teaching me the basics during my year as a technician. All of those late hours playing around with membranes and plates have paid off. And a rousing round of applause goes to Dr. Keith Ray for being so patient with all of my questions and for all of his assistance with general biochemistry and MS expertise on my project. To my dissertation committee: Drs. Rich Helm, James Mahaney, and Renee Prater. You three were extremely helpful and offered great advice and encouragement. I appreciate all of the time taken for meetings, presentations, critiques, etc. Thank you. iv I want to thank the friends I’ve made during my time in Blacksburg: Yvette Edmonds – who knew we would find our personality twin and life-long friend in the biochemistry department? The Star City Roller Girls (Roanoke, VA) – thanks for keeping me active, sane, and supplied with lasting friendships. You are a perfectly dysfunctional family for me and I wouldn’t have it any other way. And a special shout- out goes to my “derby wife” Cyndi Withrow: Thanks for being my friend, Martial Brady! The Kroenleins: I am so glad to call you family and friends; you have always been there to cheer me on and I thank you all! The Redbirds: Charlotte, Darrin and Ryan Redbird – my mom and little brothers, you have always been there whenever I call to hear my ups, downs, and everything in between. Thanks for making me who I am today and for reminding of where I come from, who I am now, and what I have yet to accomplish. I love you all very much! And finally to Karl Kroenlein, my best friend and husband: You have tolerated my love of roller derby, monkeys, cookies, Buffy, and Martha Stewart all while providing a shoulder for leaning and an ear for listening. I love the little four-legged family we have started and soon we can start adding the two-legged variety. You guessed correctly— birds. I support you always, you support me always, and that’s the way it should be. I love you the most! v Table of Contents Abstract .................................................................................................................................................... ii Acknowledgements ............................................................................................................................. iv List of Figures ........................................................................................................................................ ix List of Tables .......................................................................................................................................... xi List of Abbreviations .......................................................................................................................... xii Chapter I: Introduction ............................................................................................................................................ 1 Protein Phosphorylation Events in Living Systems .............................................................................................. 1 Protein Kinases .................................................................................................................................................................... 2 The Eukaryotic Protein Kinases or “ePKs” ............................................................................................................... 4 The Importance of Archaea ............................................................................................................................................. 7 The archaeal/eukaryotic connection .................................................................................................................... 7 Posttranslational modifications (PTMs) in Archaea ....................................................................................... 9 An Archaeal Model System for Protein Phosphorylation ................................................................................ 11 The chrenarcheon Sulfolobus solfataricus......................................................................................................... 11 SsoPK4: a serine/threonine protein kinase ..................................................................................................... 12 The Protein Kinase-Protein Substrate Problem .................................................................................................. 16 Chapter II: Materials and Methods ...................................................................................................................... 20 General Instrumentation ............................................................................................................................................... 20 Materials .............................................................................................................................................................................. 21 Materials for molecular biology ............................................................................................................................ 21 Materials for protein purification and enzyme characterization ............................................................ 22 General Methods ............................................................................................................................................................... 22 Solutions and procedures for growth of cells ................................................................................................. 22 Agarose gel electrophoresis ................................................................................................................................... 23 Cloning of open reading frames (ORFs)............................................................................................................. 23 Plasmid isolation and sequence verification ................................................................................................... 25 Site-directed mutagenesis ....................................................................................................................................... 26 Expression of recombinant
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