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Investigations of Lysine and Lysine-Derived Crosslinks in Elastin Via Solid-State Nmr Spectroscopy a Thesis Submitted to The

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Investigations of Lysine and Lysine-Derived Crosslinks in Elastin Via Solid-State Nmr Spectroscopy a Thesis Submitted to The INVESTIGATIONS OF LYSINE AND LYSINE-DERIVED CROSSLINKS IN ELASTIN VIA SOLID-STATE NMR SPECTROSCOPY A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAIʻI AT MĀNOA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN CHEMISTRY DECEMBER 2019 By Paulina Panek Thesis Committee: Kristin Kumashiro, Chairperson Philip Williams Rui Sun DEDICATION I dedicate this thesis to the people who made my journey possible. The first scientist I have ever met whose old university textbooks sparked my curiosity – my mother Grażyna Panek. My two closest friends, Patrycja Polak and Dilan Ustek, whose encouragement and support meant more than I could ever express with words. Finally, I would like to thank my very first chemistry teacher Ms. Maria Biczel, whose uplifting words were what I needed to hear to finish this thesis. ii ACKNOWLEDGEMENTS I would like to express my gratitude to my research advisor, Professor Kristin Kumashiro, for the support and mentorship she has provided for me during my graduate studies. I would also like to thank the other members of my thesis committee, Professor Philip Williams and Professor Rui Sun, for their helpful feedback and suggestions. I am grateful for the support and assistance provided by faculty members, staff, graduate students and other individuals who have helped me over the years. I am especially thankful for all the technical help provided by Dr. Kosuke Ohgo, and the camaraderie and encouragement from Dr. Chester Dabalos, Dr. Jhonsen Djajamuliadi, and David Shinsanto. Also, I would like to thank Dr. Walter Niemczura and Dr. Robert Johnson for helpful discussions. I am also appreciative of the teaching assistantships provided by the Department of Chemistry. Whether it was teaching the lab classes, or working on a mobile application for the biochemistry lab, I felt very privileged to be given the opportunity to help others in their academic journeys. iii ABSTRACT Elastin is responsible for elasticity and resiliency of force-bearing tissues in vertebrates. This polymeric protein is produced from a ~70 kDa precursor, tropoelastin, During the post-translational modification, a network of inter- and intramolecular covalent crosslinks is formed from lysine residues. As many as eleven different bi-, tri-, and tetrafunctional modifications are present in mature elastin, which are critical for normal tissue function. These products have only been characterized in hydrolysates and enzymatically-cleaved peptides, but never observed directly in the intact protein. Two isotopes of lysine, [U-13C,15N-Lys] and [13Cε-Lys], were independently incorporated into the neonatal rat smooth muscle cell (NRSMC) elastin. Samples with high levels of enrichment (~90%) were used for high-resolution solid-state nuclear magnetic resonance (ssNMR) studies. One- and two-dimensional measurements allowed for analysis of the secondary structures of lysines. Random coil and α-helices were the predominant conformations. The helical content is higher in a frozen sample than at the physiological temperature. Experiments based on cross-polarization allowed for quantification of unmodified lysine as well as desmosine and isodesmosine, crosslinks unique to elastin. The presence of rare bi- and trifunctional crosslinks was confirmed. iv TABLE OF CONTENTS DEDICATION ................................................................................................................................................. ii ACKNOWLEDGEMENTS ............................................................................................................................. iii ABSTRACT ................................................................................................................................................... iv LIST OF TABLES ........................................................................................................................................ viii LIST OF FIGURES ........................................................................................................................................ ix LIST OF ABBREVIATIONS AND SYMBOLS ............................................................................................... xi CHAPTER 1 .................................................................................................................................................. 1 INTRODUCTION ........................................................................................................................................... 1 1.1 Elastin .............................................................................................................................................. 1 1.2 Elasticity models ............................................................................................................................. 1 1.3 Biosynthesis of elastin ..................................................................................................................... 4 1.4 Crosslinking in health and disease ................................................................................................. 6 1.5 Quantification of crosslinks ............................................................................................................. 6 1.6 Crosslinking sites ............................................................................................................................ 7 1.7 Solid-state NMR .............................................................................................................................. 9 1.8 Scope of thesis .............................................................................................................................. 13 1.9 References .................................................................................................................................... 14 CHAPTER 2 ................................................................................................................................................ 17 ISOTOPIC ENRICHMENT OF LYSINES IN ELASTIN ............................................................................... 17 2.1 Isotopic enrichment via the neonatal rat smooth muscle cell (NRSMC) line ................................ 17 2.2 Targeting lysine and crosslinks ..................................................................................................... 18 2.3 Metabolism of enriched lysine ....................................................................................................... 21 2.4 Enrichment evaluation via Marfey’s chemistry .............................................................................. 24 2.5 Materials and methods .................................................................................................................. 26 2.5.1 Preparation of elastin with isotopically enriched lysine via NRSMC cultures ........................ 27 2.5.2 Quantification of isotopic incorporation .................................................................................. 30 2.6 Challenges specific to lysine enrichment evaluation .................................................................... 33 2.6.1 Low quantity of lysine and crosslinks in elastin...................................................................... 33 2.6.2 Multiple Marfey’s derivatives .................................................................................................. 35 2.6.3 Multiple isotope incorporation patterns .................................................................................. 38 2.6.4 Synergy of the challenges ...................................................................................................... 39 2.7 Results and discussion ................................................................................................................. 39 v 2.7.1 Enrichment of lysine ............................................................................................................... 39 2.7.2 Enrichment of desmosine and isodesmosine ........................................................................ 40 2.7.3 Scrambling to glycine and aspartic acid ................................................................................. 41 2.7.4 Results for remaining amino acids ......................................................................................... 42 2.8 Summary and conclusions ............................................................................................................ 42 2.9 References .................................................................................................................................... 44 CHAPTER 3 ................................................................................................................................................ 46 SECONDARY STRUCTURES AND DYNAMICS OF LYSINE RESIDUES IN ELASTIN ........................... 46 3.1 Introduction ................................................................................................................................... 46 3.1.1 Secondary structures and chemical shifts of amino acids ..................................................... 46 3.1.2 Selection of ssNMR experiments for structural studies of elastin .......................................... 49 3.1.3 Interpretation of NMR spectra with RefDB data ..................................................................... 52 3.1.4 Variable-temperature 13C MAS NMR study ..........................................................................
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