Structure and Dynamics of Elastin Cross-linking Domains by Aditi Ramesh A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Biochemistry University of Toronto Copyright c 2015 by Aditi Ramesh Abstract Structure and Dynamics of Elastin Cross-linking Domains Aditi Ramesh Master of Science Graduate Department of Biochemistry University of Toronto 2015 The secondary structure of elastin cross-linking domains has been shown to be sequence and context dependent, but the role of these domains in the function of elastomeric pro- teins remains unclear. We use molecular dynamics simulations (MD), circular dichroism spectroscopy (CD), and nuclear magnetic resonance to probe the conformational equilib- ria of model elastin-like cross-linking peptides. We tested four recently developed force fields using MD to select the one that best reproduces the amount of alpha-helix seen in CD. Simulation studies of the aggregative properties of the cross-linking domains found that they occasionally interact, but not in any specific way. Additionally, multifaceted studies of biphasic systems show that these domains do not partition preferentially into or on the interface of a hydrophobic surface. Further experiments on constructs of cross- linking and hydrophobic domains will help elucidate how cross-linking modulates the self-assembly and mechanical properties of elastomeric proteins. ii Dedication To my parents. iii Acknowledgements I would like to thank my supervisors Dr. Simon Sharpe and Dr. R´egisPom`esfor their constant guidance and advice throughout my graduate work. They have shaped the scientist I am today and fostered my deep passion for the biological sciences. I also wish to thank the members of my supervisory committee, Drs. Hue Sun Chan, Fred Keeley, and Julie Forman-Kay, for their critical analysis of my work and suggestions along the way. I wish to thank the members, both past and present, of both labs for their constant advice, help, and, most importantly, moral support. I would especially like to thank Drs. Chris Neale, Loan Huynh and Sarah Rauscher for their invaluable help in getting me started in the simulation work and teaching me the ropes. I would like to thank Zhuyi Xue for our daily discussions about elastin and Chris Ing for his scripting help in times of distress. My deepest, most heartfelt thanks to Dr. Grace Li, Kethika Kulleperuma, and Dr. Nilu Chakrabarti for their advice about everything in life. I thank Dr. Patrick Walsh and Jason Yau for leading the way in the peptide work in the Sharpe lab and teaching me the ins and outs of working with peptides for the first time. Greg Cole and Dave Davidson are thanked for their constant help and support in the lab. I have Karen Simonetti to thank for her patience, support and encouragement as I struggled in the wet lab and tried not to break equipment. I wish to thank my amazing friends Tracy Stone, Noor Alnabelseya, and countless other friends I have made over the years from my labs and the rest of the department in various labs who have kept me sane inside and outside the lab. These friends have heard me vent and cry through the years and I treasure their patience, love and support. They kept me going with their encouragement and optimism during the rough and turbulent times and have become a second family for me in Toronto. I also wish to thank Daniel Schep for his support and patience during the long months of my thesis writing. I thank with all my heart my family, especially my parents, for supporting me in all my endeavours and being the loving, encouraging people they have always been and for always having faith in my abilities, even though my own belief sometimes faltered. iv Contents List of Tables vii List of Figures viii List of Acronyms and Symbols x 1 Introduction 1 1.1 Elastin . .1 1.2 Elastin cross-linking . .5 1.3 Elastin structure and mechanism . .8 1.4 Peptide studies . .8 1.5 Recombinant elastin-like polypeptides . 10 1.6 Rationale and aims . 11 2 Methods 14 2.1 Molecular Dynamics Simulations . 14 2.1.1 Molecular mechanics . 14 2.1.2 Force fields . 17 OPLS force fields . 18 CHARMM force fields . 18 CHARMM27 (CHARMM22/CMAP) . 18 CHARMM36 . 19 CHARMM22* . 19 AMBER force fields . 20 v AMBER ff03w . 20 AMBER ff99sb*-ildn . 20 Water models . 21 Periodic boundary conditions . 21 Temperature and pressure coupling . 22 2.1.3 System setup . 22 2.2 Biophysical techniques . 23 2.2.1 Peptide synthesis . 23 2.2.2 Peptide sample preparation . 25 2.2.3 Circular dichroism . 25 2.2.4 Partitioning and analytical RP-HPLC . 25 2.2.5 NMR . 26 2.3 Data Analysis . 27 3 Results 28 3.1 Choice of force field . 28 3.2 Spectroscopic characterization of the monomeric cross-linking domains . 32 3.3 Aggregative properties of the cross-linking domains - a simulation perspective 37 3.4 Tying biophysical results back to simulation . 58 3.4.1 Solution NMR of the model peptides . 58 3.4.2 Circular dichroism spectra calculated helicity of the model peptides 60 3.5 Biphasic systems as a way to model the coacervate . 64 4 Discussion 66 5 Future Directions 74 Bibliography 76 vi List of Tables 2.1 Summary of force fields and water models used. 24 vii List of Figures 1.1 Domain architecture of the tropoelastin monomer . .3 1.2 Cross-linking domain sequences in natural elastin . .3 1.3 Pseudo-periodic hydrophobic domain sequences in natural elastin . .3 1.4 Molecular view of how cross linking is achieved in different types of elas- tomeric proteins . .6 1.5 Mechanism of cross-linking . .7 1.6 List of model peptides . 12 1.7 Position of lysines in the model peptides in a perfect α-helix . 13 2.1 Schematic illustrating the different energy terms of the potential energy formula for a force field . 16 3.1 PMFs of backbone dihedral angles of A2 in the force fields tested . 30 3.2 Time evolution of α-helix in KA16K and A7KAAKA7 ........... 31 3.3 Time evolution of secondary structure in molecular dynamics . 33 3.4 Average α-helix percentages in the A2 peptide for the four force fields tested 34 3.5 Circular dichroism spectra of model peptides in TFE . 35 3.6 Circular dichroism spectra of model peptides in NaF . 36 3.7 Circular dichroism spectra of model peptides in MeOH . 37 3.8 Average peptide-peptide distance in dimer simulations . 38 3.9 Histograms of the end-to-end distance of the model peptides in the monomer, dimer, and tetramer simulations . 40 3.10 Histograms of the radius of gyration of the model peptides in the monomer, dimer, and tetramer simulations . 41 viii 3.11 Histograms of the probability of having 0 through 18 helical residues . 42 3.12 Time evolution of the radius of gyration of A8KKA8 in CHARMM22* . 44 3.13 Time evolution of the radius of gyration for a representative A0 dimer simulation . 45 3.14 Sample contact maps for the A0 dimer system . 47 3.15 Snapshots at 100ns of six A0 dimer replicas . 48 3.16 Sample contact maps for the A0 tetramer system . 49 3.17 Snapshots at 100ns of six A0 tetramer replicas . 50 3.18 Dimer contact maps for the six model peptides averaged over all replicas 52 3.19 Tetramer contact maps for the six model peptides averaged over all replicas 53 3.20 Comparison of total fraction helix formed by all peptides over the 1, 2, and 4 peptide simulation systems . 54 3.21 Histograms of pairwise distances between the centers of mass of all peptides 54 3.22 Average fraction helicity for dimer simulations of all peptides with and without formation intermolecular contacts . 55 3.23 Average fraction helicity for tetramer simulations of all peptides with and without formation intermolecular contacts . 56 3.24 Solution NMR of the A2 peptide . 59 3.25 Secondary chemical shifts of Cα (red) and Cβ (blue) atoms for the A2, A3, and A16 peptides and A3 at different temperatures . 61 3.26 Comparison of helicity calculated from MD and CD . 62 3.27 Average helicity per residue for all the model peptides in CHARMM22* . 63 3.28 Integrated peptide peak areas from RP-HPLC chromatograms for each phase in octane and octanol partitioning experiments . 65 4.1 Position of lysines in the helical aggregated multimer simulations . 71 4.2 Schematic of the proposed cross-linking mechanism in elastin . 72 ix List of Acronyms and Symbols A˚ Angstrom AMBER assisted model building and energy refinement CD Circular dichroism DIEA N,N-Diisopropylethylamine DMF Dimethylformamide DSSP Dictionary of protein secondary structure EBP elastin-binding protein ELP elastin-like peptide EM electron microscopy Fmoc Fluorenylmethyloxycarbonyl fs femtosecond GROMACS Groningen machine for chemical simulations HFIP 1,1,1,3,3,3-Hexafluoro-2-propanol HPLC High-performance liquid chromatography K Kelvin MD molecular dynamics x nm nanometer NMR Nuclear magnetic resonance ns nanosecond OPLS optimized potentials for liquid simulations TIP3P transferable intermolecular potential function, three point model xi Chapter 1 Introduction 1.1 Elastin Elastic proteins, which are found in many animal species [1], include abductin (which is found in the flexible hinge ligament of a scallop's shell) [2], resilin (found in the cuticle of many insects) [3], spider silks [4], and elastin [5]. These proteins are found to fulfill a diverse set of functions and showcase a wide range of properties, with some spider silks demonstrating incredible rigidity while others are more elastic and resilient. The mechanical properties of elastomeric proteins motivates the study of these proteins from a biomaterials and bioengineering perspective.