Intrinsically Disordered Proteins: Mechanics, Assemblies, and Structural Transitions by Mehran Bagheri Thesis submitted to the Faculty of Graduate and Postdoctoral Studies In partial fulfillment of the requirements For the Ph.D. degree in Physics Ottawa-Carleton Institute of Physics Department of Physics, Faculty of Science University of Ottawa Ottawa, Canada c Mehran Bagheri, Ottawa, Canada, 2017 Dedicated to my loving family. To my parents, Mahin and Mohammad whose deep devotion and sacrifice enable me to follow my dreams; To my brother, Majid, for his endless encouragement in furtherance of my dreams. To my beautiful wife, Niloofar, whose continual inspiration and support keep my dreams alive and eventually turn them to reality. ii Abstract Proteins are essential parts of living organisms that initiate and control almost all cellular processes. Despite the widely accepted belief that all functional proteins fold into stable and well-defined three-dimensional (3D) structures mandatory for protein activity, the existence of biologically functional disordered proteins has been increasingly recognized during past two decades. Proteins with inherent structural disorder, commonly known as intrinsically disordered proteins (IDPs), play many roles in a biological context. However, in contrast to their folded counterparts, they are dynamically unstructured and typically fluctuate among many conformations even while performing biological functions. In fact, it is this dynamical structural heterogeneity that that allows for IDPs to interact with other biological macro- molecules in unique ways. Moreover, while a majority of proteins in eukaryotic proteomes have been found to have intrinsically disordered regions (IDR), the mechanisms by which pro- tein disorder gives rise to biological functionality is still not well understood. Through a series of simulation studies on specific systems, this thesis probes several aspects of the emerging structure-function paradigm of IDPs, namely the mechanics, intermolecular assembly, and structural transitions occurring in these proteins. The lack of well-defined 3D structure in IDPs gives rise to distinct mechanical properties, the subject of the first study in the thesis on the elasticity of a elastomeric gluten-mimetic polypeptide with an intrinsically disordered character. This disordered polypeptide was shown to exhibit distinctively variable elastic response to a wide range of tensions, which a classical worm-like chain model failed to accurately describe, thus requiring a molecular-level analysis. IDPs are frequently involved in protein-protein interactions, the focus of the second study on the propensity of an IDR, the B domain in dynamin-related protein 1 (Dpr1), to self- assemble into dimer structures while remaining disordered in all solution conditions. Despite a hypothesized auto-inhibitory role for this domain in Dpr1 that was assumed to be trig- gered by an disordered-to-order transition, the B domains in solution showed no tendency iii iv to form ordered structures even in the presence of order promoting osmolytes. Instead, self-association in the presence of osmolyte was found to occur by favorable intermolecular intereactions between specific region on the surface of the B-domains. Other IDPs do undergo a disorder-to-order transition in response to environmental cues, in ways that are unique disordered proteins, the focus of the last study on intermolecular or- dering transitions in silk-like proteins. Factors such as protein sequence and physical tension were investigated, and results suggested that tyrosine residues in the key silk sequence mo- tifs promote templating of beta structure from disordered precursors and that elongational stresses preferentialy stabilize antiparallel beta-sheet order. Together, these three computa- tional studies provide insight into the nature of the structure-function mechanisms of IDPs. Acknowledgements First, and foremost, I would like to express my sincere gratitude to my advisor Dr. James L. Harden for the opportunity to work in his laboratory. It was a great privilege to have worked and learned under the mentorship of such a knowledgeable and ambitious scientist with a great personality. Thank you for showing me nothing but patience and unparalleled professionalism over several years. I would like to thank senior scientist Dr. Ebrahim Karimi, Dr. Andrew Pelling, and Dr. Bela Joos, for continual inspiration and stimulating conversations during my graduate study. I also thank all my kind-hearted and caring colleague in the physics department, particularly Rahil Golipour and Kristina Haase. Thank you to the welcoming staff of the physics depart- ment, namely Madeleine Thomas and Lisa Murphy. Last but not least, thank you to my beautiful wife who are the source of my everlasting drive for success and without whom I could not have made it this far. Thank you, everyone. v Statement of Originality To the best of my knowledge, I hereby declare that this thesis establish original work gener- ated during my Ph.D. studies under the supervision of Dr. James L. Harden, in the Centre for Interdisciplinary Nanophysics laboratories in the Department of Physics at the University of Ottawa. Chapter4 was published whereas Chapter2,3 and AppendixA,B are prepared for peer-reviewed scientific journals. I solely generated all simulations data and perform statistical analysis to develop com- putational results while Dr. James L. Harden contributed to the direction and editing of all manuscripts. Other researchers also contributed to this work performing experiments were acknowledged in each chapter. Dr. Ammon Posey and Dr. Blake Hill introduced B domain and TMAO osmolytes explain in Chapter3. Lin Yinan and Benjamin Partlow, who were Ph.D. Candidates at the time under the supervision of Dr. David Kaplan, provided all experimental results and contributed to writing the experimental portion of the manuscript in Chapter4 and AppendixA, respectively. Python package (AppendixB) was written and developed by me; while Emilie Fortin, undergraduate summer student, edited the code and scripted a graphical user interface for the package. vi Contents Abstract iii Acknowledgementsv Statement of Originality vi Contents vii List of Figuresx List of Tables xii 1 Introduction: Functional Protein Disorder1 1.1 Principles of Protein Structure and Function..................2 1.1.1 Amino acid sequence as protein signature................2 1.1.2 Description of protein structure.....................4 1.1.3 Folding kinetics and thermodynamics..................6 1.2 Appearance of Disordered Protein........................8 1.3 Disorder Advantageous in Function....................... 11 1.3.1 Structural ensemble and entropic chain................. 11 1.3.2 Disorder interaction and regulation of protein complex........ 12 1.4 Thesis overview.................................. 13 2 Mechanics of Intrinsically Disordered Proteins 16 2.1 Background: Lessons from Polymer Physics.................. 17 2.1.1 Ideal chain and entropic spring..................... 17 2.1.2 Polymer elasticity models: worm-like chain............... 20 2.1.3 Real polymer size in solution and scaling-law.............. 22 2.2 Introduction.................................... 25 2.3 Simulation and Analysis Methods........................ 27 2.3.1 Wheat-gluten mimetic polypeptide sequence.............. 27 2.3.2 Equilibration to a compact structure.................. 27 2.3.3 Force-extension measurements...................... 28 vii CONTENTS viii 2.3.4 General simulation details........................ 28 2.3.5 Structural analysis............................ 29 2.4 Results and Discussions............................. 30 2.4.1 Structural aspects of the compact state................. 30 2.4.2 Stretching the WGM polypeptide.................... 33 2.4.3 Structural aspects impacting WGM elasticity............. 36 2.5 Concluding Remarks............................... 39 2.6 Appendix..................................... 42 2.6.1 Modified worm-like chain model fit................... 42 2.6.2 Distribution of φ dihedral angles for proline.............. 43 3 Protein Complex & Functional Disorder 44 3.1 Introduction.................................... 45 3.2 Simulation and Analysis Methods........................ 48 3.2.1 General simulation details........................ 48 3.2.2 B domain equilibration.......................... 48 3.2.3 Steered MD to construct various dimer configurations......... 49 3.2.4 Binding energy calculation for dimer.................. 50 3.2.5 Description of analysis and methods.................. 51 3.3 Results and Discussions............................. 52 3.3.1 B domain has disordered structure................... 52 3.3.2 Molecular interaction of TMAO with B-domains............ 55 3.3.3 Inter-chain associations of the B domain................ 59 3.4 Concluding Remarks............................... 64 3.5 Appendices.................................... 65 3.5.1 Forcefield Examination for TMAO molecule.............. 65 4 Disorder to Order transition in IDPs 66 4.1 Introduction.................................... 67 4.2 Simulation and Analysis Methods........................ 71 4.2.1 General simulation details........................ 71 4.2.2 Equilibration of silk solution and tyrosine self-assembly........ 72 4.2.3 Stability of silk peptide in beta-sheet structure............ 72 4.3 Results and Discussions............................. 74 4.3.1 Tyrosine colocation in solution...................... 74 4.3.2 Tyrosine residues