ENGINEERING ANTIFREEZE PROTEIN MULTIMERS THROUGH GENETIC FUSION TO SELF-ASSEMBLING PROTEIN OLIGOMERS by Sean William Phippen A thesis submitted to the Department of Biomedical and Molecular Sciences In conformity with the requirements for the degree of Master of Science Queen’s University Kingston, Ontario, Canada (August, 2017) Copyright © Sean William Phippen, 2017 Abstract Ice-binding proteins (IBPs) serve many purposes for the organisms producing them, enabling them to thrive in ice-laden environments. Antifreeze proteins (AFPs), a subset of this group, act as biological antifreezes and inhibit ice recrystallization. These ice-binding activities have many applications in the field of biotechnology, and to this end AFPs have been engineered in several ways to increase their activity. One of these strategies is to connect multiple AFPs together using a scaffold, which has been accomplished previously through bioconjugation of AFPs to the reactive termini of branched polymers. The inefficiency of such linkage reactions, however, resulted in heterogeneous mixtures of particles with incomplete occupancy of the available termini. To address this shortcoming, AFPs were genetically fused to the C termini of subunits forming self-assembling protein oligomers, in such a way that the ice-binding site of the AFP was optimally exposed for contact with ice. First, two different type of AFPs were separately fused to 12 of the 24 subunits of a self-assembling protein cage. The subunits assembled into the designed structures, and the AFP multimers showed greatly enhanced freezing point depression and ice recrystallization inhibition over the corresponding monomeric AFPs. A moderately active fish AFP was also fused to a subunit designed to assemble into two- dimensional arrays, and the resulting fusion protein showed increased freezing point depression over the monomer. However, size-exclusion chromatography of the fusion protein suggested that the subunits were not assembled into a two-dimensional array, and additional structural characterization is needed to elucidate the oligomerization state of this design. Overall, genetic fusion of AFPs to the termini of self- assembling oligomers has proven to be a promising method of enhancing their ice-binding activities, and could be used to arrange AFPs into novel orientations. We also envision that the RosettaDesign program used to design the novel protein assemblies can be applied to AFPs themselves, to generate AFP-to-AFP binding sites on their surfaces and arrange them into oligomers not seen in nature. ii Co-Authorship Chapter 2 was co-authored with Dr. Corey Stevens, Tyler Vance, Dr. Neil King, Dr. David Baker, and Dr. Peter Davies, and was published in Biochemistry on November 11th, 2016: Phippen, S. W. et al. Multivalent Display of Antifreeze Proteins by Fusion to Self- Assembling Protein Cages Enhances Ice-Binding Activities. Biochemistry 55, 6811–6820 (2016). Sean W. Phippen and Dr. Peter L. Davies planned the experimental methodology of the project. Dr. Corey A. Stevens assisted with modeling AFP multimers and with TH measurements. Tyler D.R. Vance carried out initial construct design for SfIBP, and analyzed the TH and ice plane affinity of monomeric SfIBP. Dr. Neil P. King and Dr. David Baker conceived the original design of T33-21, and Dr. Neil P. King carried out the characterization of this construct, and provided advice on the fusion with AFPs. Sean W. Phippen wrote the manuscript with guidance from Dr. Neil P. King, Dr. David Baker, and Dr. Peter L. Davies. iii Acknowledgements This work is dedicated first and foremost to my parents, Susan and Dale Phippen, who instilled in me at a young age a thirst for knowledge and taught me that I could achieve anything that I set my mind to. My brothers, Nick and Noah, are my two oldest friends and have been with me through thick and thin; thank you for sticking with me in life’s toughest moments. I would also like to thank my partner, Brigid Conroy, who has been a calming presence and stabilizing force during my time in the School of Graduate Studies. I am eternally grateful to you all for your love and support. None of this would have been possible without the mentoring of several scientists that have made me into the biochemist that I am today. Dr. Peter Davies took me on as a young, green biochemistry student and, during my time at Queen’s, has acted as an invaluable mentor and instilled in me a love of biochemistry that continues to this day. Peter has put in place an environment of innovation, teamwork, and perseverance in his laboratory, and I have enjoyed my time here immensely. Dr. Jeffrey Ridal and Jennifer Haley from the St. Lawrence Institute of Environmental Sciences gave me my first job in a laboratory, and showed me how fun research could be. I would also like to thank Drs. Virginia Walker and John Allingham for serving on my Advisory Committee and providing invaluable guidance. I am very grateful to Drs. Steven Smith, Graham Côté, and Andrew Craig for acting as members of my Examination Committee, and to Diane Sommerfeld for her tireless efforts to help me organize my coursework and this thesis. I have long known that it takes a village to raise a graduate student, and I am eternally grateful for the guidance and encouragement provided by the members of the Davies laboratory. Sherry Gauthier, Dr. Robert Campbell, Dr. Laurie Graham, Sandra Jimmo, Robert Eves, Dr. Qilu Ye, Dr. Kristen Low, Dr. Koli Basu, Dr. Shaiqui Guo, Dr. Tianjun Sun, Dr. Corey Stevens, Tyler Vance, Christian MacCartney, Evan Andrin, Paul Jerónimo, Rebecca Lang, Jigyasa Aurora, Brigid Conroy, Adam Sage, Tova Pinsky, Julia Shen, Polly Zhang, James Wells, Sarah Zachariah, and Shayan Assaie, it has been an honour and a pleasure working with you. iv Table of Contents Abstract ......................................................................................................................................................... ii Co-Authorship.............................................................................................................................................. iii Acknowledgements ...................................................................................................................................... iv List of Figures ............................................................................................................................................ viii List of Tables ................................................................................................................................................ x List of Abbreviations ................................................................................................................................... xi Chapter 1 Introduction .................................................................................................................................. 1 1.1 General Introduction ........................................................................................................................... 1 1.2 Functions of ice-binding proteins ....................................................................................................... 2 1.3 Structural diversity of IBPs ................................................................................................................. 5 1.4 Mechanism of ice-binding exhibited by IBPs ..................................................................................... 7 1.5 Ice crystal structure and shaping by IBPs ......................................................................................... 10 1.6 Applications of IBPs in biotechnology ............................................................................................. 12 1.7 Measuring AFP activity .................................................................................................................... 13 1.8 Engineering AFP activity .................................................................................................................. 16 1.9 AFP multimerization through self-assembly .................................................................................... 22 1.10 Designing novel protein assemblies ................................................................................................ 24 1.11 Protein assemblies used as scaffolds for AFP multimerization ...................................................... 27 Chapter 2 Multivalent display of antifreeze proteins by fusion to self-assembling protein cages enhances ice-binding activities ................................................................................................................................... 32 2.1 Abstract ............................................................................................................................................. 32 2.2 Introduction ....................................................................................................................................... 33 2.3 Materials and Methods ...................................................................................................................... 37 2.3.1 Design of the AFP-containing T33-21 multimers ...................................................................... 37 2.3.2 Cloning ....................................................................................................................................... 37 2.3.3 Expression and purification ......................................................................................................