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Duke University Dissertation Template Order and Disorder in Protein Biomaterial Design by Stefan Daniel Roberts Department of Biomedical Engineering Duke University Date:_______________________ Approved: ___________________________ Ashutosh Chilkoti, Supervisor ___________________________ Joel Collier ___________________________ Brenton Hoffman ___________________________ Terrence Oas ___________________________ Rohit Pappu Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biomedical Engineering in the Graduate School of Duke University 2018 ABSTRACT Order and Disorder in Protein Biomaterial Design by Stefan Daniel Roberts Department of Biomedical Engineering Duke University Date:_______________________ Approved: ___________________________ Ashutosh Chilkoti, Supervisor ___________________________ Joel Collier ___________________________ Brenton Hoffman ___________________________ Terrence Oas ___________________________ Rohit Pappu An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biomedical Engineering in the Graduate School of Duke University 2018 Copyright by Stefan Daniel Roberts 2018 Abstract Crystalline and amorphous materials have been extensively studied for their interesting properties, but they comprise a very small portion of the total materials space. The properties of most materials are a consequence of the interactions between their ordered and disordered components. This phenomenon is especially important in biology with materials such as silk and elastin owing their extraordinary attributes to the interactions of ordered and disordered domains at the inter- and intra- molecular levels. Recent insights in the emerging field of intrinsically disordered proteins have further highlighted the importance of order-disorder interactions as determinants of structural and chemical functions in multivalent proteins. While the significance of order-disorder interactions is well known and much work has been devoted to understanding their biological implications, little effort has been made to functionalize them for the development of new materials. Recombinant protein polymers offer an interesting platform for determining how combinations of order and disorder lead to unique material properties as their molecular level control enables these components to be precisely mixed within a single polypeptide chain. This dissertation reports the successful design and application of a new class of recombinant materials inspired by the protein elastin, termed partially ordered polymers (POPs), to uncover the impact of single chain interactions between ordered domains and iv disordered regions on macroscopic material properties. These ‘smart’ protein materials: (1) are the first biopolymer system with temperature dependent phase behavior in which the aggregation and dissolution temperatures can be independently controlled, (2) are injectable as a solution that assembles under the stimulus of body heat into fractal-like, porous networks suitable for cell infiltration and remodeling, and (3) can be used to create microstructures with complex architectures and spatially segregated regions for applications in drug delivery and tissue engineering. This work expands the biomedical potential for protein-based materials as well as the available microarchitectures for biocompatible polymers, demonstrating that sequence level modulation of order and disorder is an untapped principle for the design of functional biomaterials. v Dedication To my mom, for her sacrifices and never ending support. vi Contents Abstract ......................................................................................................................................... iv List of Tables ................................................................................................................................ xii List of Figures ............................................................................................................................ xiii Acknowledgements ................................................................................................................. xvii 1. Introduction ............................................................................................................................... 1 2. Background and Motivation.................................................................................................... 3 2.1 Proteins as bio-inspired materials .................................................................................. 3 2.1.1 Recombinant protein materials ................................................................................. 4 2.1.2 Stimuli-responsive protein polymers ....................................................................... 5 2.1.3 Methods for the production of recombinant protein polymers ............................ 6 2.1.3.1 Genetic synthesis .................................................................................................. 7 2.1.3.2 Recombinant expression ................................................................................... 10 2.2 Elastin-inspired biopolymers ........................................................................................ 11 2.3 Biophysical properties ................................................................................................... 13 2.3.1 Mechanism of elasticity ............................................................................................ 14 2.3.2 Phase behavior ........................................................................................................... 15 2.3.2.1 Tunability of thermal coacervation ................................................................. 18 2.3.2.2 Reversibility of thermal coacervation ............................................................. 18 2.3.3 Other biophysical properties ................................................................................... 19 2.4 Elastin-like polypeptides as models of intrinsic disorder ........................................ 20 vii 2.4.1 What is protein disorder? ......................................................................................... 21 2.4.2 Biological significance of protein disorder ............................................................ 22 2.4.3 Disorder and phase behavior ................................................................................... 23 2.4.4 Elastin: disorder at the sequence level ................................................................... 24 2.4.4.1 Hydropathy-charge ........................................................................................... 24 2.4.4.2 Tandem repeats .................................................................................................. 26 2.4.4.3 Proline and glycine ............................................................................................ 27 2.4.5 Disorder in an aggregated state .............................................................................. 28 2.5 Elastin biopolymers as injectable materials ................................................................ 29 2.5.1 Bioactive properties of elastin hydrogels ............................................................... 29 2.5.2 Mechanical properties of elastin hydrogels ........................................................... 30 2.5.3 Injectable depots for drug delivery ......................................................................... 32 2.5.4 Injectable scaffolds for tissue engineering ............................................................. 34 2.6 Summary and perspective ............................................................................................. 38 3. Stimuli-Responsive Partially Ordered Polymers ................................................................ 39 3.1 Motivation ....................................................................................................................... 39 3.2 Methods ........................................................................................................................... 40 3.2.1 Synthesis of polymer genes ...................................................................................... 40 3.2.2 Expression and purification of POPs ...................................................................... 42 3.2.3 Secondary structure characterization ..................................................................... 43 3.2.4 Temperature-dependent turbidity .......................................................................... 44 3.2.5 FRET ............................................................................................................................ 44 viii 3.2.6 Light Scattering .......................................................................................................... 45 3.3 Hysteresis in disordered biopolymers ........................................................................ 46 3.4 A library of partially ordered polymers ...................................................................... 50 3.5 Structural characterization ............................................................................................ 53 3.6 Phase behavior and tunable hysteresis........................................................................ 58 3.7 The mechanism of hysteresis .......................................................................................
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