UNIVERSITY OF CALIFORNIA, SAN DIEGO Design and Characterization of an Allosteric Metalloprotein Assembly A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Chemistry by Lewis Anthony Churchfield Committee in charge: Professor F. Akif Tezcan, Chair Professor Michael D. Burkart Professor Neal K. Devaraj Professor Tracy M. Handel Professor Valerie A. Schmidt 2018 Copyright Lewis Anthony Churchfield, 2018 All rights reserved. Signature Page This Dissertation of Lewis Anthony Churchfield is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Chair University of California, San Diego 2018 iii EPIGRAPH Epigraph Sometimes science is more art than science. Dr. Rick Sanchez iv TABLE OF CONTENTS Table of Contents Signature Page ................................................................................................................... iii Epigraph ............................................................................................................................. iv Table of Contents ................................................................................................................ v List of Abbreviations ........................................................................................................ vii List of Figures .................................................................................................................... ix List of Tables ................................................................................................................... xiii Acknowledgements ........................................................................................................... xv Vita ................................................................................................................................. xviii Abstract of the Dissertation ............................................................................................. xix Chapter 1: A survey of designed protein assemblies .......................................................... 1 1.1 Foundations of protein design: drawing inspiration from Nature ............................. 1 1.2 Repurposing native interactions for engineered protein assemblies ......................... 9 1.3 Engineering protein assemblies using de novo designed interactions .................... 16 1.4 Synergy of design strategies for native-like engineered protein assemblies .......... 27 1.5 Challenges and opportunities: emerging design paradigms protein self-assembly 30 References ..................................................................................................................... 37 Chapter 2: De novo design of an allosteric metalloprotein. .............................................. 45 2.1 Introduction ............................................................................................................. 45 2.2 Results and discussion ............................................................................................ 49 2.3 Materials and methods ............................................................................................ 70 References ..................................................................................................................... 77 v Chapter 3: Biochemical dissection of an engineered allosteric protein ............................ 80 3.1 Introduction ............................................................................................................. 80 3.2 Results and Discussion ........................................................................................... 84 3.3 Materials and Methods .......................................................................................... 116 References ................................................................................................................... 122 C38/C81/C96 Chapter 4: Molecular dynaimcs simulations of R14. ........................................ 125 4.1 Introduction ........................................................................................................... 125 4.2 Development of custom simulation parameters .................................................... 127 4.3 Molecular dynamics simulations of C38-C38 disulfide breakage. ....................... 148 4.4 Molecular dynamics simulations of C81-C81 and C96-C96 breakage. ............... 164 4.5 End-state thermodynamic analysis of C38-C38 disulfide breakage. .................... 168 4.5 Methods. ............................................................................................................... 174 References ................................................................................................................... 176 Chapter 5: Conclusion and future directions. ................................................................. 179 5.1 Concluding remarks .............................................................................................. 179 C38/C81/C96 5.2 Toward a deeper understanding of R14 allostery. .................................. 182 5.3 Exploring new elements of design complexity in the R1 scaffold. ...................... 184 5.4 Materials and Methods .......................................................................................... 191 References ................................................................................................................... 194 Appendix ......................................................................................................................... 195 vi LIST OF ABBREVIATIONS List of Abbreviations ATP adenosine triphosphate AUC analytical ultracentrifugation Csa cysteine sulfenate Cso cysteine sulfenic acid cyt b562 cytochrome b562 (contains b-type heme) cyt cb562 cytochrome cb562 (contains a c-type heme) DHFR dihydrofolate reductase DTT dithiothreitol EDTA ethylenediaminetetraacetic acid GaMD Gaussian accelerated molecular dynamics ITC isothermal titration calorimetry Kd dissociation constant LB lysogeny broth M1 Metal Binding Protein Complex 1; MBPC1 Mb Myoglobin MD Molecular dynamics MDPSA Metal Directed Protein Self-Assembly MeTIR Metal Templated Interface Redesign MOPS 3-(N-morpholino)propanesulfonic acid MTX methotrexate vii R1 Rosetta Interface Designed Construct 1; RIDC1 RhuA rhamnulose-1-phosphate aldolase Sav streptavidin SDS PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis TEM transmission electron microscopy TM apparent melting temperature TMV tobacco mosaic virus Tris tris(hydroxymethyl)aminomethane viii LIST OF FIGURES List of Figures Figure 1.1 Notable examples of naturally occurring protein assemblies. ........................... 3 Figure 1.2 Cartoon depiction of the roles of flexibility and rigidity in an artificial biological assembly. ............................................................................................................ 6 Figure 1.3 Survey of strategies for engineering protein assemblies. .................................. 8 Figure 1.4 Design of multivalent ligands for inducing protein assembly. ........................ 10 Figure 1.5 Engineering a homodimeric protein via domain swapping. ............................ 13 Figure 1.6 Design of a self-assembling protein cage by genetic fusion of oligomers. ..... 15 Figure 1.7 Design of two-component protein-DNA lattices. ............................................ 18 Figure 1.8 Designed protein scaffolds for metal-templated assembly. ............................. 20 Figure 1.9 Design of disulfide-linked protein nanorods. .................................................. 23 Figure 1.10 Designing protein-protein contacts for engineered supramolecular assemblies. ........................................................................................................................ 25 Figure 1.11 Metal tempalted interface redesign (MeTIR): a versatile strategy for protein engineering. ....................................................................................................................... 28 Figure 1.12 Emerging trends in designing protein assemblies. ........................................ 32 Figure 2.1 Successive engineering of cyt cb562 to form disulfide-linked oligomeric architectures. ..................................................................................................................... 48 II C81/C96 Figure 2.2 Structural rearrangements upon Zn removal from Zn- R14. ................. 50 Figure 2.3 SDS-PAGE gel of the products of C38/C81/C96R1 and C38/C81/C96M1 self-assembly reactions in the presence or absence of ZnII. ..................................................................... 52 C38/C81/C96 Figure 2.4 Non-reducing SDS-PAGE of R14 in the absence and presence of DTT. .................................................................................................................................. 54 C38/C81/C96 Figure 2.5 Sedimentation velocity profiles of R14. ............................................ 55 II C38/C81/C96 Figure 2.6 Binding isotherm of Zn to Fura-2 in the presence of R14. .............. 56 ix Figure 2.7 ITC thermograms of ZnII titrated into disulfide-linked R1 tetramers. ............. 58 C38/C81/C96 Figure 2.8 Overview of Zn-induced structural changes in R14. ......................... 61 Figure 2.9 Structural alignments of metal-bound disulfide-linked R1 tetramers. ............ 62 C38/C81/C96 Figure 2.10 Electron density maps of key features in R14 structures.