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Computer-Based Design of B-Sheet Containing Proteins

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Computer-Based Design of B-Sheet Containing Proteins COMPUTER-BASED DESIGN OF B-SHEET CONTAINING PROTEINS Doo Nam Kim A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry and Biophysics Chapel Hill 2016 Approved by: Brian Kuhlman Qi Zhang Andrew Lee Harold Erickson Jane Richardson © 2016 Doo Nam Kim ALL RIGHTS RESERVED ii ABSTRACT Doo Nam Kim: Computer-based Design of β-sheet Containing Proteins (Under the direction of Brian Kuhlman) Protein design is an excellent test of the minimal determinants of protein structure. Although 70% of naturally occurring proteins contain β-sheets, most previous design efforts have been limited to α-helix bundle proteins or the redesign of naturally occurring proteins. Here, we test and develop computer- based methods for designing proteins rich in β-strands. The molecular modeling program Rosetta was used for three separate design tasks: (1) the design of α/β and α+β proteins with a new method called SEWING, which builds proteins from pieces of naturally occurring proteins, (2) the stabilization of β- sheet proteins via the redesign of surface-facing residues, and (3) the de novo design of β-sandwich proteins. This research showed that it is possible to extend the SEWING method to non-α-helix proteins, allowing the incorporation of structural features found in nature, and that it is possible to dramatically boost protein thermal stability (> 25oC) with the redesign β-sheet surfaces. However, we also found that the de novo design of β-sandwich proteins still remains an elusive goal. iii “Yeah, the template file had this section.” iv ACKNOWLEDGEMENTS I deeply appreciate Brian Kuhlman, Tim Jacobs, and Bryan der for their limitless patience and teaching for me. Without them, I wouldn’t be here. I thank my family: Chanmi Choi, Philip Kim and Eugene Kim. I thank my committee members for their precious time: Jane Richardson, Harold Erickson, Andrew Lee, and Qi Zhang. I thank Nikolay Dokholyan and Qi Zhang for teaching me while I rotated. I thank all Kuhlman lab members including Alex Kenan, Alex, Alex, Amanda Loshbaugh, Andrew Leaver- fay, Andrew Lerner, Ben Stranges, Deanne Sammond, Doug Renfrew, Frank Teets, Grant Murphy, Gurkan Guntas, Hayretin Yumer, Jack Macquire, Jeffrey Jones, Jenny (Xiaozhen) Hu, Joseph Harrison, Kevin Houlihan (who gratefully taught me how to identify disulfide bonds using Rosetta), Mahmud Hussain, Matt O’Meara, Minnie Langlois, Oana Lungu, Ryan Hallet, Sharon Guffy, Steven Lewis, Stephan Kudlacek, Seth Zimmerman, and Wesley Roten. I thank Rosetta community members including Noboyasu Koga, Enrique Marcos, Gabriel Rocklin, Tom Linsky and Christopher Bahl in Baker lab. I thank my collaborators: Thomas Szyperski, Jasmin Federizon, and RamaKrishna Pulavarti. I thank my rotation students: Kisurb Choi, Devin Rodriguez. I thank Bo Zhao for this teaching. I thank UNC computing center experts: Sandeep and Jenny Williams. I thank Barry Lentz for his encouragement. I thank Ryan Lucey for his dedicated edition. v TABLE OF CONTENTS ABSTRACT ............................................................................................................................................... III ACKNOWLEDGEMENTS ....................................................................................................................... V LIST OF TABLES .................................................................................................................................... IX LIST OF FIGURES ................................................................................................................................... X LIST OF ABBREVIATIONS AND SYMBOLS .................................................................................... XI CHAPTER 1: COMPUTATIONAL PROTEIN DESIGN .................................................................... 13 Introduction .................................................................................................................................. 13 Protein as therapeutics ................................................................................................................ 13 Computational protein design .................................................................................................... 14 Challenges in computational protein design .............................................................................. 15 Necessity of de novo protein design ............................................................................................ 16 Previous de novo protein design methods .................................................................................. 17 Supersecondary structure based de novo protein design method ........................................... 20 Conclusion and following chapters ............................................................................................. 21 CHAPTER 2: COMPUTATIONAL DE NOVO DESIGN OF A/B AND A+B PROTEINS .............. 22 Introduction .................................................................................................................................. 22 Computational Design and Experimental Results .................................................................... 25 Design with three secondary structure substructures ...............................................................25 Design with five secondary structure substructures .................................................................27 Increase net charge in an effort to monomerize well-folded α+β and α/β proteins .................32 vi Reversion design in an effort to monomerize and better fold en8 design ................................34 Redesign with refolding in an effort to monomerize and better fold en8 design .....................37 Conclusion .................................................................................................................................... 38 Supporting Materials ................................................................................................................... 39 CHAPTER 3: BOOSTING STABILITY OF Β-SHEET PROTEINS BY SURFACE REDESIGN ........................................................................................... 47 Overview ....................................................................................................................................... 47 Introduction .................................................................................................................................. 48 Results ........................................................................................................................................... 52 Discussion ...................................................................................................................................... 59 Materials and Methods ................................................................................................................ 61 Computational Design and Analysis of proteins ......................................................................61 Protein Expression and Purification .........................................................................................61 Circular Dichroism ...................................................................................................................61 Fluorescence .............................................................................................................................62 CHAPTER 4: DE NOVO DESIGN EFFORTS FOR B SANDWICH PROTEINS ............................ 64 Introduction .................................................................................................................................. 64 Computational Design and Experimental Results .................................................................... 66 Design of Backbone by SEWING ............................................................................................66 Design of Backbone by Assembling β-Strands with Random Backbone Angles ....................68 Design of Backbone by ab initio Folding .................................................................................68 Design with Folding Nucleus Conservation .............................................................................69 Design with Repopulation of Existing Backbones ...................................................................69 Redesign with Native β-sandwich Backbones .........................................................................70 Conclusion .................................................................................................................................... 72 vii Supporting Materials ................................................................................................................... 72 CHAPTER 5: FUTURE DIRECTIONS ................................................................................................. 75 Towards More Efficient Protein Design .................................................................................... 75 Necessity of Experimentally Determined Structural Information .......................................... 76 Towards a More Accurate Score Function ................................................................................ 77 Towards Successful De novo Design of β-sandwich proteins ................................................... 79 Potential Applications of Designed Proteins .............................................................................
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