STRUCTURAL AND BIOPHYSICAL CHARACTERIZATION OF THE MYOCILIN OLFACTOMEDIN DOMAIN A Dissertation Presented to The Academic Faculty by Rebecca K. Donegan In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the School of Chemistry and Biochemistry at Georgia Institute of Technology Georgia Institute of Technology August 2015 Copyright 2015 by Rebecca Donegan STRUCTURAL AND BIOPHYSICAL CHARACTERIZATION OF THE MYOCILIN OLFACTOMEDIN DOMAIN Approved by: Dr. Raquel L. Lieberman, Ph.D., Advisor Dr. Adegboyega Oyelere, Ph.D. School of Chemistry and Biochemistry School of Chemistry and Biochemistry Georgia Institute of Technology Georgia Institute of Technology Dr. Ingeborg Schmidt-Krey, Ph.D. Dr. Loren Williams, Ph.D. School of Biology and School of Chemistry and Biochemistry School of Chemistry and Biochemistry Georgia Institute of Technology Georgia Institute of Technology Dr. Cheng Zhu, Ph.D. School of Biomedical Engineering Georgia Institute of Technology Date Approved: April 30, 2015 ACKNOWLEDGEMENTS First, I would like to thank my husband Brad for always supporting and encouraging me. I also wish to thank my mom and dad for encouraging me to work towards this goal since I first started college. I want to thank my siblings, grandparents, and in-laws for their constant encouragement. All of you have offered help and reassurance along the way, and I would not have reached this goal without you. I would also like to thank my lab mates, current and former, for all of their advice, instruction and help throughout the years. I would especially like to thank Dana Freeman for all of her help during her time as an undergraduate researcher. Finally, I would like to thank Dr. Raquel Lieberman, my advisor, for the opportunity to work in her lab and pursue this research. Her mentorship and guidance has helped shape me as a scientist and researcher. iii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS………………………………………………………......................iii LIST OF TABLES………………………………………………………………………………..ix LIST OF FIGURES………………………………………………………………………………x LIST OF SYMBOLS AND ABBREVIATIONS ………………………………………………..xii SUMMARY……………………………………………………………………………………...xiv CHAPTER 1. Introduction ................................................................................................................. 1 1.1 Glaucoma ............................................................................................................. 1 1.1.1 Types of Glaucoma ........................................................................................ 1 1.1.2 Genetics of Glaucoma.................................................................................... 2 1.1.3 Glaucoma and Other Diseases ...................................................................... 3 1.1.4 Aqueous Humor Production, Outflow, Intraocular Pressure and Glaucoma.... 3 1.2 Myocilin Linked with Glaucoma ............................................................................. 5 1.2.1 Steroid Induced Glaucoma ............................................................................. 6 1.2.2 Mutant Myocilin and Glaucoma ...................................................................... 7 1.2.3 Myocilin as a Potential Therapeutic Target for Glaucoma .............................. 8 1.3 Myocilin Localization, Function and Characterization ...........................................10 1.3.1 Potential Calcium Binding Site in Myocilin .....................................................11 1.3.2 Myocilin Amyloid Propensity .........................................................................11 1.4 Thesis Objectives ................................................................................................12 1.4.1 The Glaucoma-Associated Olfactomedin Domain of Myocilin is a Novel Calcium Binding Protein .........................................................................................12 iv 1.4.2 Determination of Amyloidogenic Peptide Stretches in the Myocilin Olfactomedin Domain ............................................................................................13 1.4.3 Structural Characterization of the Myocilin Olfactomedin Domain .................13 1.4.4 Structural Implications for Glaucoma Pathogenesis ......................................14 1.4.5 Identification of Ligand Binding Sites in the Myocilin Olfactomedin Domain ..14 2. The Glaucoma-Associated Olfactomedin Domain of Myocilin is a Novel Calcium Binding Protein .............................................................................................................15 2.1 Introduction ..........................................................................................................15 2.2 Results ................................................................................................................17 2.2.1 Initial identification of Calcium Stabilization in Myocilin Olfactomedin ............17 2.2.2 Elemental Analysis by Inductively Coupled Plasma Optical Emission Spectroscopy .........................................................................................................18 2.2.3 Release of Calcium upon Unfolding ..............................................................19 2.2.4 Calcium Binding Monitored by Isothermal Titration Calorimetry ....................20 2.2.5 Mutational Analysis of Carboxylic Acid-Containing Residues as Ligands for Calcium Reveals Glaucoma-Associated Aspartate 380 .........................................21 2.2.6 Structural Differences between Apo Myocilin Olfactomedin and Wild-Type Myocilin Olfactomedin ............................................................................................23 2.2.7 Investigation of Calcium Stabilization of 18 Other Disease-Causing Myocilin Olfactomedin Variants Reveals No Other Impaired Variants ..................................25 2.2.8 Estimation of Calcium Dissociation Constant ................................................27 2.2.9 Prediction of Calcium Binding Motifs in Olfactomedin Domain Family ...........28 2.3 Discussion ...........................................................................................................30 2.4 Methods ...............................................................................................................33 2.4.1 Protein Expression and Purification...............................................................33 v 2.4.2 Olfactomedin Variants ...................................................................................34 2.4.3 Thermal Stability Assay .................................................................................35 2.4.4 Fluorescence Calcium Binding Assay ...........................................................35 2.4.5 Inductively Coupled Plasma Optical Emission Spectroscopy ........................36 2.4.6 Circular Dichroism .........................................................................................36 2.4.7 Estimate of Calcium Dissociation Constant ...................................................37 3. Determination of Amyloidogenic Peptide Stretches in the Myocilin Olfactomedin Domain ..........................................................................................................................38 3.1 Introduction ..........................................................................................................38 3.2 Results ................................................................................................................41 3.2.1 Initial Amyloid Prediction in the Olfactomedin Domain of Myocilin .................41 3.2.2 Peptide Fibril Growth ....................................................................................42 3.2.3 Seeding of Full Length Myocilin Olfactomedin by Peptide Fibrils ...................44 3.3 Discussion ...........................................................................................................46 3.4 Methods ...............................................................................................................47 3.4.1 Selection and Synthesis of Peptides .............................................................47 3.4.2 Peptide Fibrillization Assays .........................................................................47 3.4.3 Peptide Seeding Assays ...............................................................................47 4. Structural Characterization of the Myocilin Olfactomedin Domain .............................49 4.1 Introduction ..........................................................................................................49 4.2 Results ................................................................................................................50 4.2.1 Crystallization of the Myocilin Olfactomedin Domain .....................................50 4.2.2 Overall Architecture of the Myocilin Olfactomedin Domain ............................53 4.2.3 Central Cavity Metal Ion and Other Identified Ligand-Binding Sites...............59 4.2.4 Comparison to Known Five-Bladed Beta-Propellers ......................................61 vi 4.2.5 Surface Electrostatics of Myocilin Olfactomedin ............................................63 4.2.6 Evolutionarily Conserved and Divergent Features of Olfactomedin Domains 63 4.3 Discussion ...........................................................................................................65 4.4 Methods ...............................................................................................................67 4.4.1 Protein Expression and Purification...............................................................67